Unexpected random urinary protein:creatinine ratio results–limitations of the pyrocatechol violet-dye method

Table 3 Dilution studies using a standard urine specimen containing 0.335 g/L of protein and either deionized water or saline as the diluent

		Measured proteinuria concentration
Dilutions of urine sample	Expected proteinuria concentrations	Water as the diluent*†	Recovery of proteinuria as a % of expected	Saline as a diluent‡	Recovery of proteinuria as a % of expected
Undiluted	0.335 g/L	n/a	n/a	n/a	n/a
1:2	0.168 g/L	0.242 g/L	144%	0.183 g/L	109%
1:3	0.112 g/L	0.199 g/L	178%	0.140 g/L	125%
1:4	0.084 g/L	0.183 g/L	219%	0.118 g/L	141%
1:5	0.067 g/L	0.172 g/L	257%	0.106 g/L	158%

n/a (not applicable).
* Water measured on its own yielded a protein concentration of 0.12 g/L.
† The unexpectedly high result for underlines the problem with dilute samples. The assay is optimized for measuring urine, not aqueous samples. Method calibrators contain protein (bovine serum albumin) added to a synthetic urine matrix with inorganic salts, a polymer, preservatives and stabilizers. Protein containing calibrators are targeted at 0.80 and 2.10 g/L and the curve is extrapolated down. The low end is verified with a calibrator at less than 0.15 g/L. In the Vitros methods, the lower analytical limit is defined by performance (precision and linearity studies) at a low level in urine or synthetic urine samples. The dye works optimally at the ionic strength of non-dilute urine (due to the various ions present), so that protein as low as 0.05 g/L can be detected with confidence. But in a low ionic strength solution (such as pure water or saline), the dye binding characteristics result in false detection of protein.
‡ Saline on its own yielded a proteinuria concentration of 0.06 g/L.

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