Influence of Freezing Temperature, Freezing Duration, and Repeated Freeze/Thaw Cycles on Electrophoretic Profiles in the White Stork (Ciconia ciconia)

Abstract

Plasma electrophoresis is an ancillary diagnostic tool in avian medicine, with agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) being the most common techniques. Frozen samples can be used for quantitative studies or comparative diagnostic purposes, but stability of avian plasma proteins under freezing is poorly described. To evaluate the influence of plasma freezing on electrophoretograms in white storks (Ciconia ciconia), heparin blood was sampled from 30 individuals during annual health examinations. Plasma samples were obtained after centrifugation of fresh samples and divided into aliquots. Both AGE and CZE were performed on fresh aliquots. The remaining aliquots were frozen at –20°C (–4°F) or –180°C (–292°F) and thawed following different protocols: 1 freeze/thaw cycle after 6 months at –20°C; 1, 2, 4, and 7 cycles over 12 months at –20°C; and 1 cycle after 18 months at –180°C. For both techniques, electrophoretic profiles obtained from these thawed aliquots were compared to fresh electrophoretograms. Quantitatively, significant differences (P < 0.05) in most fractions were seen from 6 months postfreezing at –20°C for both techniques. Fewer statistically significant differences were observed after 18 months under cryogenic preservation (–180°C). Qualitatively, AGE provided more repeatable and stable results than CZE over time on samples stored at –20°C, and electrophoretograms were stable after 18 months of cryogenic storage. An electromigration distortion associated with freezing was seen with CZE only. Plasma samples stored in a conventional freezer (–20°C) should not be compared to fresh plasma. For quantitative studies, cryogenic storage should be privileged.