Stability of osteopontin in plasma and serum.
BACKGROUND
Osteopontin is a glycoprotein widely expressed in many tissues and in different physiological conditions. Osteopontin concentrations are usually measured through immunological methods; however, little is known about the pre-analytical management of the sample. We evaluated the effects of different times and temperatures storage conditions on serum and plasma concentrations of osteopontin.


METHODS
Serum and plasma aliquots were frozen at – 80° C, following storage at 4° C or room temperature for 0, 2, 4, 8, 12, 24 and 48 h. Osteopontin concentrations were determined by enzymoimmunometric assay. Serum samples obtained from tubes with or without gel separator were compared to verify the effect of gel. Western blotting analysis was performed to characterize the antibody.


RESULTS
Osteopontin concentrations were stable over time in all conditions in both serum and plasma. Plasma showed 3.8 – 4.8-fold higher concentrations than serum. Comparable levels were found between serum tubes with or without gel separator and always lower than those in plasma, demonstrating no effect of gel in serum tubes. Western blotting analysis showed various osteopontin bands, indicating that the antibody recognizes the entire panel of different osteopontin forms.


CONCLUSIONS
We demonstrated the stability across 48 h of osteopontin in serum and plasma at either room temperature or 4° C, when the evaluation is carried out by an immune-based method. The minimal variations observed over time were always lower than the calculated intra- and inter-assay coefficients of variation. Plasma specimens should be preferred when osteopontin concentration are assayed by immunological methods.
Experimental Biochemistry and Molecular Biology Laboratory, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy. patrizia.lanteri@grupposandonato.it
Patrizia
P
Lanteri
Giovanni
G
Lombardi
Alessandra
A
Colombini
Dalila
D
Grasso
Giuseppe
G
Banfi
D000918:Antibody Specificity; D001774:Blood Chemical Analysis; D001800:Blood Specimen Collection; D015153:Blotting, Western; D004355:Drug Stability; D004356:Drug Storage; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053495:Osteopontin / Q000097:blood* / Q000276:immunology; D010949:Plasma / Q000737:chemistry*; D020033:Protein Isoforms / Q000097:blood / Q000276:immunology; D044967:Serum / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D020033:Protein Isoforms; D053495:Osteopontin