Neonates and young infants have well documented deficiencies in plasma levels of many coagulation factors (1). There is also evidence that some coagulation factors are qualitatively dysfunctional in infants (2–4). One of these coagulation factors is fibrinogen.
Speculation regarding the functional ability of the fibrinogen of infants arose from observations of prolonged thrombin and reptilase times in children (5,6). These tests measure the rate of conversion of fibrinogen to fibrin after the addition of an exogenous stimulator, either thrombin or reptilase (a proteolytic enzyme derived from snake venom) (7). Debate exists about whether these prolonged tests in infants result from the alteration of fibrin formation by extrinsic factors or from an inherent dysfunction of their fibrinogen (2,5). Extrinsic factors that can prolong at least one of these tests include heparin, pathologic serum proteins, and fibrinogen/fibrin degradation products (FDPs) (5). Although the presence of heparin or pathologic serum proteins can be and has been easily excluded, the presence of FDPs has been implicated in some cases of prolonged thrombin times (5). Other investigations, however, have found prolonged thrombin times in the documented absence of FDPs; thus indicating that the fibrinogen of infants may be inherently dysfunctional (6,8). These findings have led many investigators to propose the existence of a dysfunctional “fetal” fibrinogen in infants (2,6,9) that possibly results from the expression of a “fetal” fibrinogen gene (8).
A recent modification of thromboelastography (TEG®) provides a new method of investigating the function of fibrinogen. TEG® measures the viscoelastic properties of clotting blood and assesses all stages of clotting from initiation of coagulation through attainment of maximum clot strength and then retraction or lysis of the clot. Maximum clot strength is measured as the maximum amplitude (MA) of the TEG® tracing and is determined by the sum of the interaction of platelets and fibrinogen. This interaction occurs as fibrinogen binds to a platelet surface receptor, glycoprotein (Gp) IIb/IIIa, that is activated after the platelets have been stimulated by thrombin or by the exposed collagen of damaged blood vessels. By binding to activated Gp IIb/IIIa receptors located on different platelets, fibrinogen cross-links and aggregates the platelets (12), and the platelets orient fibrin fibers to increase their tensile strength (13). Studies in adults have shown significant consistent correlations between TEG® MA values and fibrinogen levels as well as significant, although less consistent, correlations between these MA values and platelet counts (14–17).
Addition of a Gp IIb/IIIa receptor blocker to a blood sample before obtaining a TEG® prevents this interaction between platelets and fibrinogen, abolishes the contribution of platelets to clot strength, and should result in a clot whose strength is determined only by the available fibrinogen (18). Indeed, with this TEG® modification, consistent strong correlations have been found between fibrinogen levels and the modified TEG® MA values in adults (16,17,19). This means of assessing the contributions of fibrinogen to the coagulation process has not been explored in infants and young children yet may provide insight into whether the fibrinogen of infants is functionally intact. We postulate that if the fibrinogen of infants and young children is functionally intact, then their fibrinogen levels should correlate with the TEG® MA values after the addition of a Gp IIb/IIIa receptor blocker to the blood sample as they do in adults. To test this postulate, we studied infants and young children in several age groupings to determine whether, and at what age, this correlation exists. Because fibrinogen’s actions are reflected in the TEG® α variable as well, we have also sought correlations between fibrinogen levels and the α value after adding a Gp IIb/IIIa receptor blockade to the blood sample.
The contribution of platelets to clot strength should be defined by the difference between TEG® MA values measured with and without Gp IIb/IIIa receptor blockade (ΔMA) because both fibrinogen and platelets contribute to the MA value without a blocker, whereas only fibrinogen correlates with the MA value modified with a blocker (16,20). Although correlations between platelet counts and the ΔMA have not been found to be statistically significant in adults, (16,19) correlations between platelet counts and the differences in elastic shear modulus (G), an exponential measure of clot strength derived from the MA, with and without the addition of Gp IIb/IIIa receptor blockers (ΔG), have been shown to be significant in adults (16,17). To assess the contribution of platelets to clot formation in infants and young children, we also compared platelet counts to the differences between TEG® variables obtained with and without Gp IIb/IIIa receptor blockade in this study.
Methods
After approval from our IRB, 250 children younger than 2 yr scheduled for elective cardiac surgery were enrolled to obtain 50 children in each of 5 age groups: Group I, younger than 30 days; Group II, 1–3 mo; Group III, 3–6 mo; Group IV, 6–12 mo; Group V, 12–24 mo. Written parental permission was not required.
As part of the routine preoperative laboratory screening, fibrinogen levels and platelet counts were obtained from each child the day before surgery. After the induction of general anesthesia and placement of invasive monitoring lines, a 2-mL sample of blood was drawn from the arterial line after aspirating 5 mL of blood to clear the line of heparin from the flush system. Two TEG® tracings were obtained from this blood sample. An “unmodified” tracing was obtained by immediately placing a 0.35-mL aliquot into a preheated disposable cup of a Thrombelastograph Coagulation Analyzer® (Haemoscope Corporation, Skokie, IL) containing 10 μL of 1% tissue factor (TF) to activate clotting. A tracing “modified” by the addition of the Gp IIb/IIIa receptor blocker, abciximab (ReoPro®, Centocor, Malvern, PA), was also immediately obtained by placing a 0.33-mL aliquot of this same sample into a second preheated TEG® cup containing 10 μL of 1% TF, 5 μL of abciximab (2 mg/mL), and 20 μL of 0.2 M CaCl2. The pins of the TEG® channels were raised and lowered into the cups 5 times to insure adequate mixing of the blood and additives, and a layer of mineral oil was applied to the top of the blood in each cup to prevent evaporation. The TEG® tracings were then begun and allowed to run until at least 60 min after achieving their MA. Subsequently, all tracings were analyzed by manual measurement by the same investigator (BEM).
Five values were measured from the TEG® tracings, and two indices were calculated using these measured values. The R value, measured from the beginning of the tracing until an amplitude of 2 mm is reached, represents the time necessary for initial clot formation. The K value is the time interval from the end of the R value until an amplitude of 20 mm is attained and appraises the rapidity of fibrin build-up and cross-linking as the clot forms. The α value similarly assesses this rate of clot formation; it is measured as the slope of the outside divergence of the tracing from the point of the end of the R value and reflects the function of both fibrinogen and platelets. The MA is a reflection of the maximum strength of the fibrin clot and is influenced most importantly by fibrinogen and platelets as well as by factors VIII and XIII. The A-60 value is the amplitude of the tracing 60 min after the MA has been reached and is useful in measuring clot retraction or lysis by comparing it with the MA. An A-60/MA ratio of <0.85 has been used to define fibrinolysis (21). The G = [5000]MA/[100 − MA] (15) was also calculated, as this index is an exponential, and therefore more sensitive, measure of clot strength. In every patient, each of these values and indices were obtained from TEGs® performed without (TEG®WB) and with (TEG®ABCX) the addition of abciximab.
Comparisons among the age groups of fibrinogen levels, platelet counts, and each TEG®ABCX variable were made using analysis of variance to determine whether significant differences existed among the groups. Comparisons of the individual groups were then made using two-sided Student’s t-tests assuming unequal variances with Bonferroni correction for multiple comparisons. Pearson correlation coefficients were calculated to assess the relationships between fibrinogen levels and α, MA, and G values with and without Gp IIb/IIIa receptor blockade in each age group as well as the relationships between platelet counts and α, MA, and G values without Gp IIb/IIIa receptor blockade and Δα, ΔMA, and ΔG values in each age group.
Results
The cardiac defects in the children of each age group are delineated in Table 1. The results of our measurements are compiled in the subsequent tables and expressed as mean ± sd. Table 2 shows the value of each TEG®WB and TEG®ABCX variable measured in each of the five age groups. Table 3 shows baseline fibrinogen levels and platelet counts of each age group. Platelet counts in Group I were significantly less than those in all other age groups. Group III had significantly larger platelet counts and lower fibrinogen levels than Group V.
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Table 1: Cardiac Diagnoses by Age Group
Table 2: Thromboelastogram Variables With and Without Glycoprotein IIb/IIIa Receptor Blockade with Abciximab
Table 3: Baseline Fibrinogen Levels and Platelet Counts
Correlations between fibrinogen levels and α, MA, and G values, with and without abciximab, and those between platelet counts and α, MA, and G values without abciximab and Δα, ΔMA, ΔG are shown in Table 4. The only significant correlation in Group I was between platelet counts and MAWB (r = 0.305, P = 0.031). In Group II, although fibrinogen levels correlated with αWB (r = 0.298, P = 0.036), no significant correlations were found with platelet counts. In Group III, fibrinogen levels correlated with MAWB (r = 0.520, P < 0.0001) and GWB (r = 0.556, P < 0.0001), whereas platelet counts correlated only with ΔG (r = 0.306, P = 0.031). In Group IV, fibrinogen levels again correlated with MAWB (r = 0.401, P = 0.004) and GWB (r = 0.426, P = 0.002), but platelet counts showed no significant correlations with any TEG® values. Many consistent correlations were seen in Group V: fibrinogen correlated with MAWB (r = 0.409, P = 0.003), GWB (r = 0.423, P = 0.002), αABCX (r = 0.320, P = 0.023), MAABCX (r = 0.366, P = 0.009), and GABCX (r = 0.301, P = 0.034), whereas platelet counts correlated with αWB (r = 0.493, P < 0.001), MAWB (r = 0.433, P = 0.002), GWB (r = 0.338, P = 0.016), Δα (r = 0.410, P = 0.003), ΔMA (r = 0.325, P = 0.021), and ΔG (r = 0.344, P = 0.014).
Table 4: Thromboelastogram Variables Demonstrating Significant Correlation with Fibrinogen Levels or Platelet Counts in Each Age Group
Table 5 compares the TEG®ABCX variables among the age groups. Similar comparisons for TF-activated TEG® variables measured without abciximab have already been published by our group (22). There were no differences in the RABCX values among the age groups. The KABCX, αABCX, MAABCX, and A-60ABCX values of Group II differed from those of Group V, as did the KABCX and A-60ABCX values of Group III. The KABCX value of Group II also differed from that of Group IV. There were no differences in GABCX values among age groups.
Table 5: Thromboelastogram Variables After the Addition of the Glycoprotein IIb/IIIa Receptor Blocker Abciximab
Discussion
We demonstrated that only after 12 months of age do the fibrinogen levels of young children with congenital heart disease correlate with TEG® MA values obtained after Gp IIb/IIIa receptor blockade. Therefore, we believe we have provided evidence that the fibrinogen of these infants exists in a qualitatively dysfunctional state. The multiple correlations that we found between fibrinogen levels or platelet counts and various TEG® MA and G variables in our 12–24 month age group are similar to those seen in adults (16,17,19) and may indicate a “maturing” of fibrinogen and platelets and the interaction between them at this age. Finally, the correlations between fibrinogen levels or platelets counts and TEG® α values that we found in children 12–24 months old have not been previously described in studies of adults and demonstrate the influence of fibrinogen and platelets on this TEG® variable as well.
The question of the functional competency of the fibrinogen of young children was first raised based on abnormalities of tests that measured the isolated conversion of fibrinogen to fibrin (5,6). Supportive evidence has been acquired from studies demonstrating differences in the biochemical composition and physical structure of the fibrinogen molecules of adults and newborn infants (9–11). By using TEG®, we have added evidence based on a global functional coagulation test to this debate. The discrepancy between the strong correlations found in adults between fibrinogen levels and MAABCX values and the lack of these correlations in children younger than 12 months of age in our study indicates a functional difference in the fibrinogen of adults and infants. The initial appearance of this correlation in our 12–24 month age group may define the age at which fibrinogen begins to gain functional maturity. Indeed, the appearance of multiple correlations between fibrinogen levels or platelet counts and various TEG® MA and G values in the 12–24 month age group is striking (Table 4). These correlations are in line with those found in adults (16,17,19) and may result from the fact that functionally mature fibrinogen in children 12–24 months of age is able to more appropriately interact with platelets during clot formation. The lower r values seen in these children compared with those found in adults (16,17,19), however, may indicate that this maturational process is not yet complete at 12–24 months of age.
As is the case with the MA, the TEG® α variable is influenced by the level and function of both fibrinogen and platelets (21,23). Therefore, the correlations between fibrinogen levels or platelet counts and MAWB, MAABCX, or ΔMA should also hold for αWB, αABCX, and Δα if the patients’ fibrinogen and platelets are functional. However, these correlations between fibrinogen levels or platelet counts and α values were not assessed in the studies on adults (16,17,19). We have found that correlations between fibrinogen levels and αABCX, and between platelet counts and Δα do, indeed, exist, but only in the 12–24 month age group (Table 4). This is in accordance with the correlations found with MA values (Table 4) and strengthens our conclusion that fibrinogen is functionally mature only after 12 months of age, and emphasizes the importance of fibrinogen and platelets in determining the α value.
Interestingly, the α, MA, and G values of our unmodified TEGs® in children <12 months old seem unaffected by fibrinogen that we are claiming is qualitatively dysfunctional (Table 2). An explanation for this may lie in the fact that platelets and fibrinogen have been shown to compensate for one another to produce normal TEGs® MA values when one of the two is deficient in number or function (16). The TEG® in our study were activated using TF to allow rapid attainment of MA values. The use of TF maximally activates platelets, causing them to significantly augment fibrin clot strength (18). Thus, the unmodified TEG® values of the infants in our study most likely remain “normal” even in the presence of the dysfunctional fibrinogen of infants because of the compensatory effect of maximally activated platelets.
Our findings may help explain why certain transfusion practices are necessary and effective in infants after cardiopulmonary bypass (CPB). The use of cryoprecipitate in this situation has been championed by several authors because of the low fibrinogen levels found in infants at this time and because of the improvement in TEG® values and postoperative chest tube drainage that result from its administration (24,25). Although the efficacy of cryoprecipitate has been accredited to its restoration of fibrinogen levels, it may also be important that the transfused fibrinogen in the cryoprecipitate is obtained from adult donors and, therefore, is composed of functionally mature fibrinogen that augments the qualitatively deficient fibrinogen of the infants that remains after CPB. This qualitative advantage may supplement the quantitative restoration of fibrinogen levels accomplished by the administration of cryoprecipitate.
Our data may also be useful in the future planning of algorithms for managing post-CPB coagulopathies in children. The use of TEG® with and without Gp IIb/IIIa receptor blockade may help differentiate the need for platelets or cryoprecipitate to restore hemostasis. However, because correlations among fibrinogen, platelets, and TEG® variables do not hold in children <12 months of age, modified TEG® will not be useful in differentiating whether platelets or cryoprecipitate are most specifically needed at a given time in infants.
In conclusion, our data obtained from TEGs® modified by Gp IIb/IIIa receptor blockade indicates that the fibrinogen of infants with congenital heart defects exists in a dysfunctional state. The appearance in children 12–24 months of age of correlations between fibrinogen levels or platelet counts and various TEG® variables that are similar to the correlations found in adults indicate a “maturing” of fibrinogen, platelets, and the interactions between them at this age. The strength of the correlations, however, may indicate that the maturation process is not yet complete at this age. These findings add more information to the ever-evolving study of the coagulation status of infants and young children and may provide evidence to justify and guide our management of the coagulopathies that follow CPB in these children.
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