Leger, Rachel

Abstract Introduction: The prevalence of venous thrombosis and pulmonary embolism (VTE) increases with age. Definitive VTE diagnosis or exclusion relies on imaging modalities, which are found to be negative in a high proportion of tested patients. Algorithms based on pretest clinical probability (low and intermediate) in conjunction with negative d-dimer test results have reliably excluded VTE sparing the need for additional imaging studies. Currently, the cutoff for a negative d-dimer is based on reference ranges regardless of age of the donor population. Since baseline d-dimer increases with aging, an age-adjusted d-dimer (AADD) cutoff for patients older than 50 years (age*10 ng/mL FEU) has been shown to improve specificity of diagnosis while still maintaining a high sensitivity (>97%) of detecting VTE/PE (Righini, et al. JAMA, 2014). Due to FDA restrictions, clinical laboratories cannot implement and report such AADD cutoffs. Major hurdles include: AADD cutoffs are assay-dependent, and there is a lack of feasible strategy for laboratories to verify the AADD cutoffs. We hypothesize that an adequate study of age-distribution of baseline d-dimer levels of healthy donors can assist the verification of AADD cutoffs and ultimately ensure patient safety in the VTE patient population. Objective: Establish an age dependent reference range for patients 50 years or older with the HemosIL HS 500 D-dimer reagent on the ACL TOP 700 (Instrumentation Laboratory, Bedford MA, USA); and compare this range with the manufacturer provided and literature recommended AADD cutoffs. Methods: D-dimer assay was performed on plasma samples from normal adult donors per the manufacturers' instructions. D-Dimer age, gender distribution and association were analyzed using non-parametric Wilcoxon rank sum, Kolmogorov-Smirnov tests, and quantile regression methods to estimate the 95th percentile to establish a reference range. Results: We studied 241 healthy donors, ranging from 21-91 years of age. Statistically significant differences between males and females (100 [42%] male) were detected (p-value <0.001; p-value =0.0009). Using CLSI guidelines, these statistical differences (median d-dimer for men = 200, while median d-dimer for women = 300) are not considered clinically significant, since only 6% of women would be outside an overall reference range (CLSI guideline states to partition if over 8% for either group would be outside the overall reference range). Using the quantile regression method, a significant association between d-dimer and age was found at the median (P<0.0001). We then established a reference range (95th percentile) for donors younger than age 50 at 635 ng/mL FEU for both men and women and AADD reference range for patients 50 and older (635 + (age - 50)*10 ng/mL) (Figure 1). This reference range was verified with our sample set, with only 6.2% of donors higher than the established reference limit. If we used the non-AADD cutoff provided by the manufacturer for donors younger than 50 (500 ng/mL FEU) and the literature recommended AADD cutoffs for older donors (age*10 ng/mL FEU), 13.3% of our normal donors were above the limits, resulting in not validating this literature reference range per CLSI guidelines in EP28-A3c. These data imply that if both manufacturer and literature recommended AADD cutoffs were adopted, HemosIL HS 500 D-dimer assay should have sufficient sensitivity and negative predictive value, although with slightly lower specificity than those reported by Righini, et al. for detecting VTE/PE. Conclusion: Our study verifies the association between d-dimer level and age and for the first time, to our knowledge, determined AADD normal range for the HemosIL HS 500 D-dimer assay. The manufacturer and literature recommended AADD cutoffs should provide sufficient sensitivity and negative predictive value for VTE/PE exclusion. Figure 1 Age distribution of D-Dimer values (n=241). Figure 1. Age distribution of D-Dimer values (n=241). Disclosures No relevant conflicts of interest to declare.

Abstract Background: Aortic stenosis-associated acquired von Willebrand syndrome (AS-AVWS) is caused by an accelerated degradation of the highest molecular weight von Willebrand factor (VWF) multimers (HMWM). We recently reported that the severity of the HMWM loss correlates with the severity of AS and patients’ bleeding tendency (Am J Cardiol, 2013;111:374-81). As for the missing HMWM, it is still uncertain if they are cleared from circulation or redistributed with medium-low MWMs, which can be assessed by VWF propeptide (Pro)/antigen (Ag) ratio. Besides the tedious VWF multimer analysis, it is uncertain if VWF activity by immunoturbidic method (Lx)/ Ag ratio or VWF collagen binding activity (CBA)/Ag ratio can be used as a screening test to predict AS-AVWS. It has been speculated in the literature that VWF:CBA is more sensitive than VWF:Lx for detecting a HMWM loss. Our goal was to exam the laboratory characteristics of various VWF tests in evaluating AS-AVWS and the potential mechanism of HMWM loss. Method: Sixty-six patients (between years 2010-2012, 43% male) with varying degrees of AS (16 mild, 20 moderate, and 30 severe) and separate 21 patients who had aortic valve replacement were assessed with VWF:Ag, VWF:Lx, VWF:CBA, platelet function analyzer collagen plus adenosine diphosphate (PFA-CADP), VWF multimer analysis and multimer ratio by densitometry (Am J Cardiol, 2013;111:374-81) after their echocardiography. The clinical endpoints of this study are AS severity measured by mean gradient (MG) by echocardiography and clinically significant bleeding. Statistical analyses including Spearman rank correlation test, estimation of the area under the receiver operating characteristics (ROC) curve, and Wilcoxon rank sum tests were performed. Results: VWF:Lx and VWF:CBA had strong correlation (Spearman r=0.94, P<0.001), and the correlation of VWF:CBA/Ag and VWF:Ltx/Ag was 0.49, p<0.001). As previously published, in patients with AS, MG correlated with VWF:Lx/Ag (Spearman r = -0.41, p <0.001), PFA-CADP (r = 0.49, p <0.001), and VWF multimer ratio (r = -0.76, p <0.001). MG also correlated with VWF:CBA/Ag (r= -0.38, p=0.002), but not with VWF: Pro/Ag (r=-0.20, p=0.12). Among AS and AVR patients, the area under the ROC curve for detection of the loss of HMWM was 0.88 (95% CI: 0.80-0.95) by VWF multimer ratio, 0.76 (95% CI: 0.65-0.86) by PFA-CADP, 0.70 (95% CI: 0.58-0.82) by VWF:CBA/Ag ratio, 0.67 (95% CI: 0.56-0.70) by VWF:Lx/Ag ratio, and 0.53 (95% CI: 0.41-0.65) by VWF:Pro/Ag . In addition to previously published results, clinically significant bleeding, in 14% of AS patients, was not associated with VWF:CBA/Ag (p=0.25) or VWF:Pro/Ag ratio (p=0.83). VWF: CBA/Ag ratio was significantly lower in patients with severe AS (MG > 40 mmHg) compared to AVR patients (median: 0.84 vs. 1.06, p<0.001), while there was no evidence of a difference in VWF:Pro/Ag (p=0.45). Conclusion: HMWM losses are highly prevalent in patients with moderate to severe AS. Of the VWF tests, PFA-CADP and VWF multimer analysis and ratio had the highest predictive ability for AS-AVWS followed by VWF:CBA/Ag and VWF:Lx/Ag. The VWF:CBA is not overly superior than VWF:Lx for predicting a HMWM loss. The normal VWF:Pro/Ag suggests that AS-AVWS is different from other types of AVWS due to accelerated clearance of HMWM. Disclosures No relevant conflicts of interest to declare.