(A) Spiking experiments showed that luciferase luminescence is a reliable detection method for protein expression in blood, since hemoglobin interference proved problematic with colorimetric detection. Luminescence signal was detected in an ECL imager (Thermo Fisher Scientific, Rockford, IL, USA) for different lengths of time with auto-gain. We tested different lysing methods. Lysate-IA represents the supernatant fraction obtained after lysis with nitrogen cavitation method. Lysate 1B was the corresponding pellet re-suspended in extraction buffer. Lysate-II was obtained by lysing the cells with hypotonic solution containing PBS. Lysate-III was obtained by shearing the cell suspension by passing through an 18-gauge needle. Only blood lysate-IA showed luminescence with an increasing dose response. (B) Lysates made from the same source but using two different methods were tested along with blanks (no DNA). All samples received substrate followed by imaging. Nluc signal was detected in Lysate-IA (supernatant of nitrogen cavitation method) but not in either blanks or Lysate-IB (the pellet of nitrogen cavitation method was resuspended in extraction buffer and tested for Nluc expression). (C) Protease inhibitors (P.I.) were tested to see if expression would improve and was found to increase the Nluc activity about 2 to 3 fold when compared with no P.I. 1 ×, 2 × and 3 × represents Nluc substrate concentration. (D) Reproducibility of Nluc expression in leukocyte cell-free system. Nluc was expressed with and without RNase inhibitor. Luciferase analysis using Nluc substrate analyzed on ECL imager.