In our pragmatic study of primarily outpatient women attending high-risk pregnancy clinics, we found that the random urinary PrCr was inflated when urinary creatinine concentration was <3 mM, regardless of pregnancy outcome. This relationship was indeed surprising, as taking a ratio of protein and creatinine concentrations should correct for urinary dilution.
In seeking an explanation for increased PrCr results at low urinary creatinine concentrations, we considered both physiological and analytical explanations. First, we saw inflated random urinary PrCr results when analyses were restricted to women with urine samples with SG >1.010 or those with no dipstick hematuria or leukocytes. Second, considering that proteinuria in pregnancy is measured primarily to detect pre-eclampsia, either de novo or superimposed on pre-existing hypertension (present in 15% of our cohort), it must be noted that women with pre-eclampsia tend to be intravascularly volume contracted so if anything, they should have decreased urine volumes (or even oliguria) and elevated (not decreased) urinary creatinine concentrations. Many of these women are placed on bedrest, but this would not account for decreased urinary creatinine concentration because the natural history of pre-eclampsia is too short to have a significant impact on muscle mass (and urinary creatinine excretion); women at term gestations are delivered right away  and those remote from term are delivered for maternal/fetal reasons either right away (40%) or on average, within 10–14 days (30%) . Third, in considering the role of analytical methods, in two other patient cohorts from our centre (which used pyrogallol red dye-based protein assays until 2009, and Vitros thereafter) random urinary PrCr results were inflated in samples tested by the Vitros compared to those tested using pyrogallol red. Of particular note, 10% of 24-hour urine samples were dilute  and thus, subject to the same problem as the random urine samples in the current study. Finally, dilution studies showed that using our pyrocatechol violet molybdate dye-binding method on the Vitros analyser, there was overestimation of urine protein as dilution increased, and even pure water and saline contained ‘measurable’ protein. This led us to the conclusion that our random urinary PrCr results were inflated in dilute urines because of falsely high urine protein results.
These results helped to explain both our clinical observations that some women with elevated random urinary PrCr results had normal pregnancy outcome, as well as the findings of a previous report of unexplained proteinuria of 0.38 g/d associated with high water intake (and low 24-hour urinary creatinine concentration of 2.9 mM) in 63 people in a community screening study . In that published study, the Vitros 950 autoanalyser was used to measure protein, and when 56 of the subjects agreed to decrease their water intake, urine protein significantly decreased (to 0.16 g/d, associated with an increase in urinary creatinine concentration to 6.9 mM).
The manufacturer of the Vitros urine protein assay (Ortho-Clinical Diagnostics, Rochester, NY) has documented that when urinary SG is ≤1.010, measurement of 24-hour urine protein excretion may be falsely elevated as a limitation of the assay . We found, however, that urine creatinine <3 mM was more effective than urine SG ≤1.010 for identifying dilute samples in which random urinary PrCr was overestimated; the inaccuracy of urine dipstick SG compared to other measures of urine concentration is well documented , whereas the use of creatinine to correct for variability in urine concentration is the premise of PrCr assessment . The overestimation is attributed to the fact that the accuracy of pyrocatechol violet molybdate dye-binding relies on the ionic strength of the urine. Lower ionic strength (as seen in dilute urine) may result in more dye-binding and higher measured protein concentrations . This overestimation by Vitros of protein concentration in dilute urine has been previously published [11, 12]. Consistent with the manufacturer’s explanation was our observation that when saline was used as the diluent in our dilution studies (thus, offering some ionic strength from the sodium chloride), inflation of urinary protein measurement seen with use of deionised water was attenuated. Although this study showed that proteinuria results were inflated in dilute urine because of analytical bias, it is to be noted that the use of creatinine may also fail to correct for urinary dilution in very dilute urines for other analytes, for example, as demonstrated for urinary albumin .
Our 2008 review of the diagnostic accuracy of the random urinary PrCr concluded that it is a “reasonable” rule-out test for detecting proteinuria of ≥0.3 g/d in hypertensive pregnancy . Our findings in the current study should have no impact on this conclusion. Although that review could not examine the impact of urine protein assay method on the results because of incomplete reporting of methods, none of the included studies used the current Vitros method; five used the dye pyrogallol red.
It should be highlighted that this is not a problem restricted to random urinary samples; 10% of 24-hour urine collections may be dilute (i.e., have urinary creatinine <3 mM)  and proteinuria is assayed in 24-hour urine samples by taking a sample, measuring the urine protein concentration, and then multiplying it by the volume of urine submitted to get the value for protein excretion in g/d.
The impact on maternal management of a false positive proteinuria result may include patient anxiety and greater use of health care resources due to enhanced maternal and fetal surveillance (as a diagnosis of pre-eclampsia is associated with both maternal and perinatal risk), hospital admission, and/or earlier delivery . Higher costs related to misclassification of renal function related to creatinine standardization have also been previously published .
The first strength of our study is that in our current study cohort, we maximised generalisability by assessing a broad spectrum of (primarily outpatient) high-risk patients with and without significant proteinuria as diagnosed by a random urinary PrCr of ≥30 mg/mmol. We also expanded our analyses to two other cohorts of primarily inpatient women with pre-eclampsia. Second, we performed an additional exploratory dilution study using the Vitros proteinuria method, and validated our result in two separate cohorts of women with proteinuria measurement by a different dye-based method (i.e., pyrogallol red).
Some would point out that the random urinary PrCr result was not compared to 24-hour urinary protein; however, it must be acknowledged that the 24-hour urine collection is frequently incomplete in pregnancy and as such, is no longer considered to be the gold standard for diagnosis of proteinuria in pregnancy .