We investigated sample collection variables that may influence the measurement of the thyroid stimulating hormone (TSH) including: time after birth, season, different ways for blood spot drying and varied elution time from filter paper. TSH was measured with an enzyme-linked immunoassay (EIA) method on dried blood spots collected from newborns and/or external quality control materials from CDC. We found that TSH results were stable if specimens were collected from newborns 72 hours after birth. We obtained different results when TSH was measured during different seasons. The results also changed as the specimens were dried in different ways. The length of time for eluting from the DBS also exerted influence on the TSH measurement. In order to assure newborn screening quality, all factors influencing the results should be considered and the best condition for testing chosen. The specimen should be collected from babies at 3-6 days of age and air-dried at room temperature. Different cut-offs may be necessary for different seasons of the years.