Characterizations of urinary sediments precipitated after freezing and their effects on urinary protein and chemical analyses

Abstract

One of the obstacles in analyzing frozen urine samples is the formation of uncharacterized precipitates. Frequently, these precipitates are discarded before analysis. Some laboratory data may be erroneous if these precipitates contain important compounds. In the present study, we examined urinary sediments precipitated after overnight storage at -20 degrees C. Although cells and debris were removed before freezing, the precipitates remained, whereas storing the centrifuged urine overnight at 4 degrees C did not result in precipitate formation. There were no significant differences observed among 10 healthy individuals (5 men and 5 women). EDTA (5 mM) could efficiently reduce the amount of precipitates to approximately 25% of the initial amount. The addition of exogenous CaCl2, but not sodium oxalate and NaCl, significantly increased the amount of precipitates in a dose-dependent manner. Linear regression analysis revealed a significant correlation between endogenous urinary calcium level and the amount of precipitates (r = 0.894; P < 0.001). Urine pH also had some effects on the type and amount of precipitates. These precipitates were composed mainly of calcium oxalate dihydrate and amorphous calcium crystals. The results also showed that these precipitates could deplete urinary proteins and calcium ions (23.6 +/- 1.1% decrease). Therefore, these freezer-induced urinary sediments significantly affect protein analysis and measurement of calcium levels in the urine. However, vigorous shaking of the sample at room temperature could redissolve these precipitates. Our data strongly indicate that these freezer-induced precipitates must be taken into account when the frozen urine samples are analyzed.

PCR-ready human DNA extraction from urine samples using magnetic nanoparticles

“…Despite considerable efforts to extract gDNA from urine sediments, traditional gDNA extraction methods are easily contaminated by various known enzyme inhibitors (phenol, urea and salts) originating from the urine and/or extraction process. Consequently, it would be necessary to re-dissolve the sediment prior to cell isolation, as adopted by protocols assaying other urine components (e.g., proteins) [26]. Despite reports that incubating urine samples at high temperatures [9] or room temperature with vigorous agitation [26] could re-dissolve the sediments, these approaches were unsatisfactory in our experience, particularly when the urine samples had a high sediment content.…”

“…The urine used in this study however, was freshly prepared, but in many applications, urine samples need to be stored at low temperatures before processing. Low temperature storage is problematic as sediment may form within the urine, particularly under frozen conditions [26], which contains many physiological metabolites that may inhibit enzymes if not removed completely. The presence of sediment also strongly interferes with urine cell gDNA recovery using magnetic particles as solid-phase adsorbents.…”

Serum creatinine and cystatin C provide conflicting evidence of acute kidney injury following acute ingestion of potassium permanganate and oxalic acid

“…Since these studies were carried out in two hospitals where laboratory facilities are limited, we were not able to examine for oxalate crystals in the urine samples immediately after collection. It is not valid to examine crystals in thawed stored samples [63].…”

A study on the effect of the internal exposure to 210Po on the excretion of urinary proteins in rats

“…Urine bio-specimens stored at low temperature (-20 to -80°C) are known to form a precipitate (mainly composed of calcium oxalate dihydrate) after thawing to room temperature (Saetun et al 2009). In addition to calcium ions, these precipitates have been shown to deplete urinary proteins during storage at -20°C in as short as 12 h (Vittinghus 1990).…”