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We have established the assay conditions of midkine (MK) measurement, the reference intervals and evaluation for clinical significance of blood MK measurement. MK is a kind of cytokines and basic protein which is a heparin-binding growth factor of various cells. The increase of MK expression suggests a prognostic value in early stage on cancers or inflammation. But significant problems in the MK measurement are alterations resulting from standing time and temperature instability after blood collection. Assay of MK was performed with solid phase human MK immunoassay recently developed sensitive enzyme linked immunosorbent method. The assay condition of MK was required to be separated immediately after blood sampling within 24 hrs at 4 degrees C or within 2 hrs at room temperature-standing. Plasma sample obtained with EDTA-2Na or citric acid-Na, and serum obtained from plain tube container showed good results. Linearity was obtained up to 1500 pg/ml and repeatability and reproducibility were within 10% as CV%. The recovery of MK was 101.1+/-3.8% with 10 specimens ranged 97-105%. Addition of interfering substances showed no effect on assay results when hemoglobin, EDTA-Na, citrate and turbidity check, but conjugated bilirubin (over 0.68 mmol/l) and gave negative errors within 10% in the assay results and heparin gave negative errors. The reference interval was 550 +/- 160 pg/ml in healthy individuals serum.