Evaluation of storage and handling conditions

The quality of data obtained from any study will be limited by the quality of the specimens used and thus, the integrity of samples is an important consideration. To delineate and identify the changes that can occur to specimens as a result of differences in storage conditions, a study was designed to systematically test the effects of storage under various time and temperature conditions (Tab. 3).

SELDI-TOF analysis was performed on these timed samples and results are displayed in Tab. 4. Although there were no major differences observed between storage at — 20°C, — 80°C, and liquid nitrogen, over the 2 month time period as detected by SELDI-TOF under our conditions, there were multiple peak differences noted that were present or absent at both room temperature and refrigerated storage

Fig. 1 Differential peptide display of plasma and serum specimens from African American donors. (A) This heat map indicates differences between plasma (blue) and serum (red). (B) This panel depicts peptides, which are generated by thrombin and plasma kallikrein during coagulation of serum samples in a time dependant manner (15 min to 8 h). The graph illustrates the kinetics of generation and degradation of the activation peptide of coagulation factor XIII (AP-FXIII). Time kinetics have been carried out in separate experiments at BioVisioN. (C) This panel depicts a zoom view into mass spectra representing a distinct chromatographic area and m/z region. Four lanes from peptide displays (African American (AfA) and Asian American (AsA)) serum specimens are shown. The AP-FXIII is solely displayed in serum samples. The additional peak in AfA samples (arrow) represents the Leu34 polymorphism of AP-FXIII.

Fig. 1 Differential peptide display of plasma and serum specimens from African American donors. (A) This heat map indicates differences between plasma (blue) and serum (red). (B) This panel depicts peptides, which are generated by thrombin and plasma kallikrein during coagulation of serum samples in a time dependant manner (15 min to 8 h). The graph illustrates the kinetics of generation and degradation of the activation peptide of coagulation factor XIII (AP-FXIII). Time kinetics have been carried out in separate experiments at BioVisioN. (C) This panel depicts a zoom view into mass spectra representing a distinct chromatographic area and m/z region. Four lanes from peptide displays (African American (AfA) and Asian American (AsA)) serum specimens are shown. The AP-FXIII is solely displayed in serum samples. The additional peak in AfA samples (arrow) represents the Leu34 polymorphism of AP-FXIII.

(Tab. 4). A subset of these samples, i.e., those that had been incubated for 30 days at each tested temperature, were selected for more in depth investigation using SDS-PAGE (Fig. 2) and 2-DE (Fig. 3). There were a number of protein bands on 1-D SDS gels that decreased or appeared, presumably relating to proteolytic fragments and/ or modifications, after long term storage at higher temperatures. Similarly, specific protein spots decreased or appeared on 2-D gels (Fig. 3) under the various time and temperature conditions.

To facilitate correlation of these results to more standard clinical testing, the levels of fourteen protein analytes in serum were also measured (Supplementary Tab. 2). A number of alterations in protein levels and enzyme activities under the various conditions tested were noted. The results demonstrate that there are changes in enzyme activity and protein stability for several proteins: lactate dehydrogenase (LDH), aspartate-amino transferase (AST), and lipase show decreases at room temperature over the 2-month period. Amylase and alanine-amino transferase (ALT) show changes at both room temperature and — 20°C storage, during the same time period.

Tab. 3 Time and temperature conditions used for stability testing. Serum samples were prepared and stored under the appropriate condition for the requisite time period. Samples were then denatured and stored until testing was initiated (see text for details)

R/T (230C) Refrigerated (40C) Frozen at -20oC Frozen at -80oC Liquid nitrogen

0h 2h

4h 4h

30d 30d 30d 30d 30d

60d 60d 60d

Interestingly, alkaline phosphatase activity decreased by storage at — 20°C, but was not altered by storage at room temperature. It was also noted that two of the cholesterol components, LDL and HDL, showed increases by extended storage at room temperature.

Was this article helpful?

0 0

Post a comment