and Stroke – Preliminary Results of a Case-Control Study
R. Schobess, W. Kreuz, K. Kurnik, C. Duering and U. Nowak-Göttl
Summary
Elevated factor (F) VIIIC: concentrations are associated with an increased risk of venous thrombosis in adults. To evaluate the role of persistent elevated FVIII:C-Ag in children with venous thrombosis (VT) or ischemic stroke (IS) the present study was performed. FVIII:C-Ag concentrations (Asserachrom VIIIC:Ag, Diagnostica Stago) were measured along with established prothrombotic risk factors six to twelve months after the acute thrombotic onset in 171 white children aged neonate to 18 years suffering a first VT (n = 69) or IS (n =102) and in 255 healthy controls.
The cut-off values defined as age-dependent 90thpercentiles were obtained from the 255 healthy children. The hereditary nature of elevated FVIII:C-Ag was confirmed in family studies. Median (range) values of FVIII:C-Ag were significantly elevated in children with VT compared with controls [117(25-374)IU/dl vs. 96(21-192)IU/dl;
Mann-Whitney tied p-value < 0.000], but not in children with IS [95(49-192)IU/dl vs. 96(21-192)IU/dl; p = 0.7]. In addition, when comparing FVIII:C-Ag in subjects above the 90thage-dependent percentiles with children below the cut-off logistic regression adjusted for age and the presence of established prothrombotic risk factors (factor V G1691A, factor II G20210A, elevated lipoprotein (a), deficiency sta- tes of protein C, S and antithrombin, elevated antiphospholipid antibodies) showed a significantly increased odds ratio (OR) and 95% confidence interval (CI) in child- ren with VT [OR/CI: 2.9/1.4-6.1; p = 0.004] but not in patients with IS [1.8/0.9-3.6;
p = 0.11]. Data shown here give evidence that familiarly elevated FVIII:C-Ag con- centrations independently increase the risk of VT in children 2.9-fold. In contrast, elevated FVIII:C-Ag levels did not play a significant role in pediatric IS so far.
Introduction
Venous and arterial thrombosis are rare diseases that are being increasingly dia- gnosed and recognized also in infancy and childhood. Due to the special properties of the hemostatic system during infancy and childhood, symptomatic thrombotic manifestation occurs in 0.07/10,000 children, 5.3/10,000 admissions of children, and 2.4/1000 admissions of newborns to intensive care units [1-4]. Within the entire childhood population, possibly due to the lower concentrations of antithrombin, heparin cofactor II and protein C along with a reduced fibrinolytic capacity, neona-
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34thHemophilia Symposium Hamburg 2003
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tes are at a greater risk of thromboembolic complications than older children. The incidence of vascular accidents decreases significantly after the first year of life, with a second peak during puberty and adolescence again associated with reduced fibrinolytic activity [1, 2]. Numerous clinical and environmental conditions, such as peripartal asphyxia, neonatal infections, fetal diabetes, the use of central lines, trau- ma or surgery, dehydration, malignant diseases, renal diseases, autoimmune disea- ses, or the intake of oral contraceptives by adolescent girls resulted in elevated thrombin generation with subsequent thrombus formation in infancy and child- hood [2-7]. In addition to the stated underlying clinical conditions, various genetic prothrombotic defects, particularly those affecting the physiological anticoagulant systems, i.e. antithrombin, protein C and protein S deficiency, the mutation of coa- gulation factor V (G1691A), and the prothrombin gene variant (G20210A), have been well established as risk factors for thrombotic events [8]. In addition, metabo- lic diseases such as homozygous homocysteinuria and moderate hyperhomocystein- emia as well as increased concentrations of lipoprotein (a), have recently been shown to significantly enhance the risk of thromboembolic arterial and venous thrombosis in pediatric and adult patients [9-13]. The association of multiple hemostatic prothrombotic defects or the combination of established prothrombo- tic risk factors with acquired environmental or clinical conditions greatly increases the risk of thrombosis, not only in adults but also in infants and children [11].
Several studies have been demonstrated that elevated factor VIIIC: concentra- tions are associated with an increased risk of venous thrombosis in young and elderly adults [14-20]. Thus, to evaluate the role of persistent elevated FVIII:C-Ag in children with venous thrombosis (VT) or ischemic stroke (IS) the present study was performed.
Materials and Methods Ethics
The present study was performed in accordance with the ethical standards laid down in the updated relevant version of the Declaration of Helsinki and approved by the medical ethics committee at the Westfälische Wilhelms-University, Münster, Germany.
Study Period
1018 pediatric patients aged neonate to 18 years with a first symptomatic throm- boembolic event were consecutively recruited and screened for hereditary pro- thrombotic risk factors between January 1995 and June 2003 by the following par- ticipating centres in the catchment areas of Hamburg, Kiel, Lübeck, Münster, Bielefeld, Düsseldorf, Berlin, Magdeburg, Halle and Munich [9, 11, 12].
Inclusion Criteria
Inclusion criteria were a thromboembolic event confirmed objectively by standard imaging methods, i.e., duplex sonography, venography, CT or MR imaging for the diagnosis of venous thromboembolism, and cerebral CT scanning, MR imaging, MR angiography, or transcranial Doppler ultrasonography for the diagnosis of thromboembolic ischemic stroke.
Exclusion Criteria
Patients older than 18 years at onset, children not of Caucasian origin, patients with incomplete clinical or laboratory work-up (established prothrombotic risk factors), and subjects lost for follow-up or without parental consent were not enrolled in the present study.
In addition, children during the acute thromboembolic phase, patients with renal insufficiency, liver disease or malignancies, subjects on heparin therapy, and adolescent girls taking oral contraceptives were excluded.
Final Study Population
From the ongoing multicenter study, a subgroup of 171 white patients with a medi- an (range) age of 6.4 (0.6 to 18) years was analyzed. Thromboembolic manifestati- on included VT (n=69) and thromboembolic IS (n=102).
Control Group
Patients were compared with 255 white infants from different geographic areas of Germany recruited between January 1996 and June 2003. These control infants, who had no history of chronic disease or of thromboembolic events and were not on medication at the time of recruitment, had presented as outpatients for evaluation before minor surgery (planned circumcisions and hernias) or bone marrow dona- tion [9, 11, 12].
Laboratory Tests
With informed parental consent, the factor V G1691A (FV) and factor II G20210A (FII) mutations, concentration of lipoprotein (Lp) (a), protein C (PC), protein S (PS) and antithrombin (AT) levels were investigated in patients and controls 6-12 months after the acute event [16], using standard laboratory techniques [9, 11, 12].
Factor VIIIC:Ag was investigated with a commercially available ELISA (Assera- chrom VIIIC:Ag, Diagnostica Stago). Since data on normal values for factor VIIIC:Ag in children are sparse, FVIII:C-Ag concentrations > age-dependent 90th
percentiles derived from the healthy control children were used as cut-off values.
A type I deficiency (antithrombin, protein C) was diagnosed when functional plasma activity and immunological antigen concentrations of a protein were repeat- edly shown to be below 50% of the normal age-related limit. A type II deficiency (antithrombin, protein C) was diagnosed in patients with repeatedly low functional activity along with normal antigen concentrations. The diagnosis of protein S defi- ciency was based on reduced free protein S antigen levels combined with decreased or normal total protein S antigen concentrations respectively. Criteria for the he- reditary nature of a hemostatic defect were its presence in at least one first degree family member, or the identification of a causative gene mutation, or both.
Statistical analysis
All statistical analyses were performed with the StatView 5 software package (SAS Institute) and the MedCalc software package (MedCalc, Mariakerke, Belgium).
Prevalence rates of prothrombotic risk factors in patients and control subjects were calculated by c2-analysis or by Fisher’s exact test. To compare the overall rate of pro- thrombotic risk factors between patients and controls and to evaluate the interac- tion between decreased FVIII:C-Ag concentrations and established prothrombotic risk factors, odds ratios (ORs) together with 95% confidence intervals (CIs) were estimated from a multivariate analysis using a logistic regression model. The signi- ficance level was set at 0.05. For variables with a non-Gaussian frequency distri- bution, data were presented as medians and ranges. All evaluations and compari- sons between patients and controls were conducted using the Mann-Whitney test (p-values < 0.05 were considered significant).
Results
Age-dependent Distribution of FVIII:C-Ag
The upper age-specific 90thpercentile was 113 IU/dl in children aged 3-6 months, 142 IU/dl in children from 6.1 to 12 months, 138 IU/dl in subjects from 1-9 years, and 148 IU/dl in subjects aged 9.1 to 18 years.
Median/Range FVIII:C-Ag Concentration in Patients and Controls
The median (range) level of total FVIII:C-Ag was 117 IU/dl (25-374) in the children with VT compared with 96 IU/dl (21-192: p < 0.0001) in the controls. Children with IS showed no elevated FVIII:C-Ag values 95 IU/dl (49-192; p= 0.7).
Logistic Regression Analysis
In the multivariate analysis, including the overall rate of established prothrombotic risk factors known to be significantly associated with symptomatic thromboembo- lism in pediatric patients in Germany, e.g. the FV G1691A mutation, the FII G20210A variant, deficiency states of protein C, S or antithrombin, or elevated Lp(a) concentrations, elevated levels of FVIII:C-Ag > the age-specific 90th percentiles retained their statistically significant and independent association in children with venous thrombosis (OR/CI 2.9/1.4-6.1; p=0.004). No such association was found for the stroke children tested (OR/CI 1.8/0.9-3.6; p=0.11). In children with persistent elevated FVIII:C-Ag levels the inheritance of low concentrations was confirmed in at least one family member [18].
Discussion
The importance of various hereditary hemostatic abnormalities in contributing to the risk of pediatric thromboembolism is well established [8]. To date, the rate of single or combined prothrombotic risk factors detected in Caucasian children is approximately 50%, while no thrombophilia has been found in the remaining subjects [9-12]. Confirming the findings of adult patients with venous thrombosis [14-20], data presented here give evidence that elevated FVIII:C-Ag concentrations
> age dependent 90thpercentiles further contribute as a significant and indepen- dent risk factor to symptomatic venous thrombosis in Caucasian children [21]. The OR found was 2.9 for children with venous thrombosis, i.e. clearly within the range reported for further inherited thrombophilic risk factors in white children, e.g. the heterozygous FV G1691A mutation, the FII G20210A variant and elevated lipo- protein (a) levels.
In summary, besides the established risk factors, a FVIII:C-Ag concentration
> 90thage-dependent percentiles is another risk factor for venous thrombosis in Caucasian children. However, the findings of the present study are restricted to white German patients, and further studies are recommended to clarify the role of elevated FVIII:C-Ag in pediatric populations not of Caucasian origin.
Acknowledgement. The authors thank Susan Griesbach for help in editing this manuscript. This study was supported by an unrestricted grant awarded by the Karl Bröcker Stiftung.
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