Time-dependent decline in D-dimer immunoreactivity in dried serum stains: a preliminary one-year longitudinal study using a latex agglutination assay.

BACKGROUND: D-dimer is used forensically to identify blood and to distinguish menstrual or postmortem blood from peripheral blood. How D-dimer immunoreactivity changes as blood dries and ages is largely unknown, despite its relevance to bloodstains of uncertain age. Since DNA and proteins degrade differently, we addressed this experimentally rather than extrapolating from DNA degradation. METHODS: Serum from nine medicolegal autopsies was diluted to ten concentrations (100-1000 μg/mL) and spotted onto filter paper as dried serum spots, stored indoors at 22 °C. Spots were tested monthly for 12 months with a latex agglutination kit (RAPIDIA®-D-dimer II), starting at the lowest concentration and advancing when the previous one tested negative; three examiners judged agglutination by stereomicroscopy. As an exploratory supplement, residual 100 μg/mL spots from three samples were quantified with a multiplex ELISA platform (Ella) at five time points. RESULTS: The detection threshold rose steadily in all nine samples, from 111.1 to 455.6 μg/mL over 12 months (∼4.1-fold; R2 = 0.9891), corresponding to a decline in detectable D-dimer to roughly one-quarter of baseline. Examiners agreed at every time point. The exploratory RFU data declined more steeply within the first two months, then more gradually, loosely compatible with a power-law curve and tentatively suggesting D-dimer could remain detectable longer than a linear model predicts. CONCLUSIONS: D-dimer remained detectable in dried serum stains for at least one year, but immunoreactivity declined progressively. The staircase assay and exploratory ELISA data suggest different, not necessarily incompatible, decay shapes, which may inform semiquantitative estimation of bloodstain age pending larger studies.