The results for unmanipulated samples and for samples

at pH 6.5, 7.3, and 8.0 are summarized in Fig. 1. Intermediate results were seen at pH 7.0 and 7.6 (not shown). Immediately after the pH manipulation, the b-crosslaps increased 0.2–6.6% (P .0.05). After 2 days, all samples exhibited a significant decrease of the b-crosslaps concentrations. In the control samples, b-crosslaps decreased by 12% at 37 °C and by 5% at room temperature on the second day of storage. After 5 days at room temperature, the decreases in the b-crosslaps concentrations in the samples were even more pronounced. The b-crosslaps concentrations in the samples stored in the refrigerator remained unchanged. In the pH-manipulated aliquots, we observed a positive correlation between the pH and the b-crosslaps concentration (Fig. 1). The lower the serum pH, the lower was the measured concentration of b-crosslaps. This pH-dependent decrease in the b-crosslaps concentration occurred more rapidly at higher storage temperatures. In the 37 °C samples, the decreases at pH 6.5 and 8.0 were 57% and 13%, respectively. The changes in the room temperature samples were similar, but less pronounced, after 2 days. After 5 days at room temperature, the decreases at pH 6.5 and 8.0 were 64% and 7%, respectively. At 4 °C, the decreases in b-crosslaps were 6–36% after 5 days and 8–54% after 10 days. The pH-induced effects were in addition to the changes caused by the various storage procedures. The explanation for the observed decreases in b-crosslaps is not clear. It may, in part, reflect a pH-dependent change of conformation in the octapeptide, from the b-form into the isomeric a-form, which is not detected by the antibodies of the test. Furthermore, it is not clear whether a switch from the dto the l-enantiomeric form is of any importance in this assay. Another explanation could be a degradation of the octapeptide or the detecting antibody by proteolytic enzymes in serum with different pH and temperature optima. We conclude that inappropriate preanalytical treatment can significantly complicate clinical interpretation and that standardized preanalytical treatment is necessary. At a serum pH .8.0, the b-crosslaps concentration remains relatively stable for some days if stored in a refrigerator. We recommend centrifugation of the blood samples immediately after their arrival in the laboratory and storage at 4 °C (refrigerator) for no more than 5 days.