Deep vein thrombosis (DVT) remains one of the major vascular diseases causing morbidity and mortality worldwide¹. Assessment of the age of the thrombi to be treated is of clinical significance because thrombolytic agents not only are ineffective in treating aged and organized clots but also can cause severe side effects². While various imaging methods have been reported^3-5, there still is an urgent need for a directly translatable and quantitative technique for evaluating the age of thrombus.A murine complete stasis model of inferior vena cava (IVC) thrombosis was established as described previously⁶. In brief, under anesthesia, BALB/C mice (male, six-week-old, n=6) received a midline laparotomy to expose the infrarenal portion of the IVC, followed by IVC ligation using 7-0 non-reactive prolene sutures. At different time points (1, 7, 14, and 28 days) from the time of stasis ligation, IVC segments containing thrombi were collected and fixed in 10% formalin overnight, then transferred to PBS and examined by MRI, followed by histological assessment. Ex vivo MRI was conducted on a vertical bore 11.7 T Bruker Avance imaging system equipped with a 15 mm volume RF coil. MT-MRI scan was conducted using a modified RARE (TR/effective TE = 6000/4ms, RARE factor=8, two slices, slice thickness=1mm, FOV=14x14 mm², matrix size=128 x 128, resolution=0.1 x 0.1 mm², and 2 averages) pre-saturated by a CW RF pulse (4 s and 4.7 μT) at 18 offsets ranging from 1 to 20 KHz (2.5 to 50 ppm). In addition, a T1 map was acquired at the exact same geometry and spatial resolution using a RARE-VTR sequence (effective TE=25 ms and RARE factor=4) with 12 TR times ranging from 60 ms to 10 sec. Data processing was performed with custom-written scripts in MATLAB using the two-pool Super-Lorentzian model⁷. In the present study, we hypothesized that the age of clots is correlated with their macromolecular content, which can be determined using the quantitative Magnetization transfer (qMT) technique (Figure 1). The acquired qMT data were fitted using a two-pool exchange model to extract the macromolecule fraction (f) or bound pool fraction (BPF), and the transfer rate between the macromolecules and water (R) and the T2 relaxation times of free water (T2a) and macromolecules (T2b). Figure 2b shows the parametric maps of thrombi collected at different times after stasis. As shown in Figure 2c, the MT data acquired using a 4.7 µT CW pulse fitted the two-pool Super-Lorentzian model described by Ramani et al⁷ very well. Consistent with previous study^8,9, all thrombi showed an elevated MTR (Figure 2a), reflecting the accumulation of macromolecules including fibrin and collagen. Among the parameters estimated from qMT data, macromolecule fraction (f) was found to be markedly increased in aged thrombi (28 days) as compared to those in the acute phase (1- 14 days), namely from ~ 20% to ~50% (Figure 2d). In some areas, the exchange rate (RM0b) also increased in the aged thrombi (28 days), but appeared less sensitive and more heterogeneous. T2 relaxation times of free water (T2a) and macromolecules (T2b) didn’t show correlation with age of thrombi. Unlike conventional MTw images, the proposed qMT approach is capable of measuring the pathology-relevant parameters macromolecule fraction, f, and transfer rate between the macromolecules and water, R, which are independent of MRI acquisition parameters. Our ex vivo studies clearly demonstrate the feasibility of using qMT to quantify the amount and organization of large macromolecules in thrombi at different time points after stasis. Our next step will be to apply this qMT method for in vivo measurements and to monitor treatment response.We have tested the ability of using Quantitative Magnetization transfer (qMT) technique to stage clots based on their macromolecular content. Data acquired on ex vivo thrombi samples clearly showed that macromolecule fraction (f ) can be used as a useful parameter for distinguishing organized blood clots from freshly formed ones accurately, and hence is indicative of thrombus aging.