On effect can be introduced in various ways. We found that if a coagulation enzyme, e.g., FXIa or FVIIa, is added manually to multiple wells on a microtiter plate, a wellto-well drift appears due to the different durations of contact between PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 the enzyme and plasma inhibitors. A similar effect may be observed if TF or contact activator is added manually before recalicification. In our experience, long exposure of plasma to fluorogenic substrate prior to recalcification also decreases the TGT response. Edge effects can be Boc-D-Lys-OH caused by uneven heating of microplate inside the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 reader and artifacts of liquid dispenser. It should be noted that internal normalization on the thrombin-2macroglobulin activity calibrator (e.g., as proposed by Hemker et al. [21]) is not helpful here because the thrombin calibrator only corrects for fluorogenic signal-related inaccuracies and fails to correct for biological and pipetting variables [22]. All location effects may be corrected with the use of the strip-plot sample design. Specifically, when edge artifacts are minor, our symmetrical strip-plot designLiang et al. Thrombosis Journal 2013, 11:12 http://www.thrombosisjournal.com/content/11/1/Page 8 ofThrombin peak height (nM)AStandard (calibration) curve location1 2 3 4 5 6 7 8 9 10 11A B C D E F G HBFitted standard curve300 250 200 150 100 50 1 10Column 1 Columns 1 2 average polynomial fitTCTCFactor XIa (pM)CIndividual columns Symmetrically averged columnsFactor XIa activity calculated against standard curve (pM)200 1 2 3 4 5 6 7 8 9 10 11Column #Figure 6 Biases introduced by location artifacts in the commercial CAT instrument. A. Plate layout demonstrating two locations of calibrations curves, column 1 vs. averaged symmetrical columns 1 + 12, used for bioassay analysis in this figure. "TC" denotes thrombin calibrator wells. B. Calibration curves from column 1 (filled symbols) and averaged columns 1 + 12 (open symbols) fitted using polynomial equations (red lines). Means ?S.D. (n = 2 wells). C. Calculated FXIa concentrations obtained for either individual columns 1 through 12 against calibration curve from column 1 (filled symbols) or for averaged symmetrical columns 1 2-(2-Aminoethoxy)-5-chloropyridine hydrochloride + 12, 2 + 11, etc. against the calibration curve from columns 1 + 12. The activities of FXIa samples were calculated under the assumption that each column contained a single sample that was serially diluted, starting with the highest "neat" sample in the first well of the respective column, followed with 2.5 dilutions for wells 2 through 7 (well 8 was "blank" buffer control). In the bioassay, TPH of each well in the column series was compared with the calibration curve (Figure 6B), and resultant FXIa concentration was multiplied by the respective dilution factor. Thereafter, the FXIa data from the first 6 wells were averaged, and S.D. was calculated. The highest diluted sample in well 7 was ignored because it was too close to the limit of assay detection.provides the simplest practical solution for microplatebased TGT experiments.Competing interests The authors declare that they have no competing interests. Authors' contributions Dr. MVO designed the study, developed thrombin generation analysis software and wrote the paper. Dr. YL and SAW conducted the experiments presented in this paper. Dr. AMS conducted preliminary experiments and assisted with method development. Dr. TKL contributed to the study interpretations and helped writing the paper. All author's read and approved the final manuscript.