Measurement of immunoglobulin free light chains in serum.

but the active/latent isoform proportions were similar (Fig. 1A). Platelet aggregation during clotting (6) may have caused these differences. MMP expression was lower in K 2 E plasma than in LH plasma [14 (3) vs 25 (4) ␮g/L; P Ͻ0.05; Fig. 1A, lane 4 vs lane 5]. The concentrations of MMP-9 forms decreased significantly with increasing amounts of K 2 E during PB collection, whereas MMP-2 was increased (P Ͻ0.01; Fig. 1B). When we added anticoagulants to the zymography buffer (to mimic the conditions in Vacutainer Tubes), only K 2 E inhibited the gelatinolytic activities (data not shown). Although EDTA may alter MMP expression (7), the reasons for the contrasting K 2 E effects remain unknown. To minimize interindividual variability , we collected PB from the same individual into different buffers. We found mainly proMMP-2 in the buffered/acidic citrate plasma [202 (15) ␮g/L], whereas there were no statistically significant differences among the 9NC, ACD, and CPDA plasmas. We found additional proMMP-9 in the K 2 E, LH, and NaF/ KOx plasmas (Fig. 1A, lanes 1–3 vs lanes 4 – 6). Our observations revealed that anticoagulants can act as preana-lytical determinants of PB MMPs. LH and 9NC plasmas collected after Lympholyte gradient (Fig. 1C, lanes 3 and 4 vs lanes 1 and 2), as well as after 9.65% sodium diatrizoate alone (Fig. 1C, lanes 5 and 6 vs lanes 3 and 4), showed increased concentrations of all MMPs. Polysucrose 400 alone did not affect MMP concentrations (data not shown) or isoform profiles. Cytometric analysis revealed differences in MMP composition between leukocytes from LH PB vs leukocytes from 9NC PB (data not shown). Physiologic buffy coats from 9NC PB showed only MMP-9 forms, had lower gelatinase activity, and had a different zymographic profile with respect to LH PB (Fig. 1C, lane 7 vs lane 8). The MMP differences between LH vs 9NC plasma could be caused by differential release of MMPs from, e.g., platelets and leu-kocytes, with a changed MMP con-tent/profile depending on the anti-coagulant used (6, 8). Although previous reports suggested heparin as the anticoagulant of choice to study circulating MMPs (2, 3), to optimize the diagnostic validity of PB MMPs as cancer biomarkers (1), we recommend the use of buff-ered/acidic citrate (9NC, ACD, and CPDA), whereas LH, K 2 E, and NaF/ KOx, which affect the MMP content and zymographic profiles of plasma and leukocytes, should be avoided. specimen collection …