late samples. Age standardised detection and false positive rates for different screening protocols were produced. Results: We collected two blood samples in 27 pregnancies affected by trisomy 21 and in 3891 control pregnancies. The early samples were taken between gestational week 8+0 up to week 13+6, and the late samples between week 11+3 up to week 14+6. The median interval between the samples was 17 days (range 1–40 days). We found a significantly better (P < 0.05) estimated screening performance when using early sampling vs. late sampling. With a risk cut-off of 1 in 100, at the time of the risk assessment, the estimated detection and false positive rates were 91% (95% CI: 81–98%) and 1.6% (95% CI: 1.3–2.0%) respectively. Better estimated performance was achieved with the use of the double set of markers; detection rate 93% (95% CI: 85–99%) and false positive rate 1.7% (95% CI 1.4–2.0%), but this was not significantly different from the early sample protocol (P > 0.05). Conclusions: Using early sampling with measurement of PAPPA and free β-hCG in the combined first trimester screening can optimise screening performance for trisomy 21. Using a double set of the maternal serum markers has the potential to further improve screening performance.