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Warfarin dosing relies on accurate measurements of international normalized ratio (INR), which is calculated from the prothrombin time (PT), International Sensitivity Index international sensitivity index (ISI) of the thromboplastin, and the geometric mean of normal PT (MNPT). However, ISI assignments of certain reagent/instrument combinations are frequently unavailable, especially when the reagent and instrument are not from the same manufacturer. The effort to be in compliance with widely endorsed Clinical and Laboratory Standards Institute (CLSI) guidelines by locally verifying or assigning an ISI to an unsupported reagent/instrument combination is further hindered by the lack of US Food and Drug Administration (FDA)-approved certified plasmas designated for a particular reagent/instrument combination. The objectives of the study include development of a process to verify/assign ISI and MNPT of a single thromboplastin reagent from one manufacturer across multiple instruments including several from another manufacturer and across several campuses of a single institution, the Mayo Clinic. In this study, RecombiPlasTin 2G (R2G), was evaluated on the ACL TOP 700 (IL), STA-R Evolution, STA Compact, and STA Satellite. Random normal donor samples (n = 25) were used to verify/assign MNPT. A subset of the normal donors (n = 8) and 13 warfarin pools (INR range: 1.3-3.9), created from stable warfarin patient plasma, were used for ISI verification/assignment. The manufacturer's assigned ISI was first verified on the ACL TOP 700 (reference method), then assigned on three unsupported instruments using orthogonal regression analysis. The MNPT and manufacturer assigned ISI (11.0, 0.95) were verified on the ACL TOP 700 and subsequently assigned on the STA-R Evolution (11.6, 1.04); STA Compact (11.5, 1.02); and STA Satellite (10.9, 0.99). Linear correlations of the INR results from all the four instruments demonstrated an r2 > 0.99. This process provides a reproducible approach to assigning ISIs on unsupported reagent/instrument combinations. Our data also confirm that ISIs of the same PT reagent differ significantly on different instruments, thus confirming the requirement for evaluations and validation of ISIs for different reagent/instrument combinations.

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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.