Urine is a liquid waste product secreted by the kidneys, consisting primarily of 91 to 96 percent water. The remaining solution contains dissolved organic compounds like urea and creatinine, and inorganic salts such as sodium, chloride, and potassium. Freezing a urine sample is common for preservation, but the process fundamentally alters the sample’s physical and chemical state. While integrity remains stable for some analyses, freezing often introduces changes that can skew results for other types of testing.
Reasons for Sample Preservation
Freezing urine samples stabilizes biological and chemical components for delayed analysis. In medical diagnostics, samples are frozen if testing cannot be completed within the standard 24-hour refrigeration period. Laboratories often hold specimens for retrospective or confirmation analysis, requiring them to be maintained in a stable state for weeks or months.
Freezing is prevalent in research and biobanking, where large cohorts are collected for long-term studies, such as metabolomics. These studies require reliable storage, often for many years, to allow researchers to analyze biomarkers or track changes. In forensic or legal contexts, samples are frozen to ensure the chain of custody and integrity until final legal review or secondary testing. Storage at ultra-low temperatures, such as -80°C, effectively halts the degradation of sensitive compounds like hormones, proteins, and nucleic acids.
Chemical and Physical Changes from Freezing
The freezing process introduces physical and chemical instability to the urine matrix. Physically, ice crystal formation can damage or rupture cellular components present in the sample, such as red blood cells, white blood cells, and epithelial cells. This cell damage, known as lysis, releases intracellular contents, which can inaccurately elevate measured values or make microscopic examination unreliable.
Chemically, the most significant change is the precipitation of dissolved solutes, which occurs as water freezes first, concentrating the remaining salts. These precipitated solids are often composed of amorphous calcium crystals and calcium oxalate dihydrate. This precipitation significantly reduces the concentration of certain ions, like calcium, and can trap larger molecules, such as proteins like uromodulin and albumin, within the sediment.
The loss of these molecules into an insoluble precipitate can lead to falsely low readings for tests measuring protein or calcium levels. The method of freezing can also alter the sample’s pH, especially if dry ice is used, as sublimating carbon dioxide introduces carbonic acid. Although most metabolites remain stable through one freeze-thaw cycle, these shifts mean frozen samples are less reliable for certain routine urinalysis components than fresh or refrigerated samples.
Proper Handling for Freezing and Thawing
To minimize degradation, urine should be collected in a sterile, airtight container, ideally durable plastic to prevent breakage upon expansion during freezing. The sample should be frozen immediately after collection, preferably within a few hours, to limit bacterial growth and chemical breakdown. For long-term storage, temperatures of -20°C are acceptable, but -80°C is preferred by research facilities to ensure the stability of sensitive analytes.
Proper thawing is important to restore the sample’s homogeneity before testing. Slow thawing is the most common practice, involving placing the sample in a refrigerator overnight or allowing it to warm gradually at room temperature. Rapid heating methods, such as microwaving, should be avoided as they can degrade heat-sensitive components and damage the container.
Once thawed, the sample often contains visible white precipitates or sediment due to salt and protein fallout. Before analysis, the sample must be thoroughly mixed, typically using a vortex mixer or vigorous shaking, to re-suspend these solids and ensure the test aliquot is representative of the entire sample. Minimizing the number of freeze-thaw cycles is recommended, as repeated cycling increases the risk of cell lysis and further precipitation, compromising the sample’s integrity.