A total of 200 clear, transparent sera without red pigmentation (low hemoglobin content) were first selected by visual screening from the total sera available in the database. Hemoglobin content of each serum sample was then determined according to [40] with minor modifications. A calibration curve was generated using five standard solutions (concentrations: 1.8, 3.6, 5.4, 7.2, and 9 μg/ml) of porcine hemoglobin diluted in 400 μL commercially available porcine serum (Sigma Aldrich, St Louis, MO, USA). Triplicate samples were incubated for 5Ámin at RT, then absorbance (E) was measured at 380, 415, and 440 nm. Absorbance at 380 and 440 nm was used to discern background absorbance flanking the absorbance peak (415Ánm) of oxygenated hemoglobin. Absorbance due to hemoglobin was calculated as: E415-[(E380 + E440)/2]. Hemoglobin absorbance values of the samples were converted to μg/mL of hemoglobin by means of the calibration curve. Of the 200 initial samples, a total of 120 samples having an absorbance ≤ 0.085 (corresponding to a hemoglobin content below 4.52 μg/mL) were included in the study; 50 in the discovery and 70 in the validation phases, respectively. Viral RNA extraction from the sera was performed following standard Roche procedures (High Pure Viral RNA Kit, Roche Diagnostics GmbH, Germany). Presence or absence of PRRSV was determined by multiplex PCR of conserved regions of viral ORF7 using primers and conditions previously described [41, 42] . The test also enabled to discriminate European and American genotypes and could detect all the different viral strains present in the Lombardy region at the time of sample collection.