Objectives: This study sought to determine the correlation between protein-to-creatinine ratio and 24-hour urinary protein excretion, to examine agreement between the 2 methods, and to determine the discriminant value for protein-to-creatinine ratio that reliably determines significant threshold levels of proteinuria.

Materials and Methods: Proteinuria was assessed by 24-hour urine protein excretion and protein-to-creatinine ratio. Correlation and limits of agreement between the 2 methods were evaluated. The discriminant cutoff values for spot urine protein-to-creatinine ratio in predicting 24-hour urine protein excretion were determined using receiver operating characteristic curves.

Results: A positive correlation (r=0.7459, P < .0001) was found between spot urine protein-to-creatinine ratio and 24-hour urine protein excretion. A Bland-Altman plot shows that the 2 tests have reasonable limits of agreement at a low level of protein excretion, but the limits become wider as protein excretion increases. The area under the receiver operating characteristic curve for urine protein-to-creatinine ratio at various cutoffs was 0.967 (95% confidence interval: 0.880-0.996; P < .0001). The cutoff level of 0.433 had a sensitivity and specificity of 100% and 90%.

Conclusions: We conclude that the protein-to-creatinine ratio in spot urine specimens in patients undergoing a kidney transplant is a convenient and reliable method of estimating protein excretion in urine.

Key words : Proteinuria, Correlation, Agreement, Jordan

Introduction

Measurement of protein excretion in a 24-hour urinary collection (UP) is the criterion standard for the quantitative evaluation of proteinuria. An alternative method for quantitative evaluation of proteinuria is to measure the protein-to-creatinine ratio (PCR) in an untimed spot urine specimen, which provides a more convenient method of assessing protein excretion and is recommended by National Kidney Foundation Kidney Disease Outcomes Quality Initiative guidelines.¹ Although there is moderate to high correlation between PCR and UP,^2-7 agreement should be assessed when considering replacing one technique with another.⁸ Few studies have tested the agreement between these 2 urinary protein measurement techniques,^{9- 11} with varying results.

In the present study, the correlation between PCR and UP, agreement between the 2 methods, concordance analysis, and a discriminant value for PCR that reliably determines significant threshold level of proteinuria will be determined.

Materials and Methods

Sixty-nine outpatients with positive dipstick for protein, attending the nephrology outpatient clinic at Jordan University Hospital, were evaluated. The protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration. Written, informed consent was obtained from all patients. Although all patients were given clear instructions how to collect the urine accurately, 10 patients were excluded because the UP was either inadequate or overcollected. The remaining 59 patients (43 men and 16 women) were included. Their characteristics are shown in Table 1. The specimens of 24-hour UPs and random urine specimens were collected within 2 days of each other.

The concentration of total protein in urine was measured by turbidimetric assay using Benzethonium chloride, and the urine creatinine was measured by a creatinine Jaffe test using Roche/Hitachi 917 analyzer (Roche Diagnostics AG, Industriestrasse 7, CH-6343 Rotkreuz, Switzerland).

Statistical analyses
Analyses of data were performed using MedCalc statistical software (MedCalc Software, version 12.3.0; Ostend, Belgium). Spearman’s rank correlation coefficient between the spot PCR and 24-hour UP was calculated. The limits of agreement between the 2 methods were analyzed by the Bland-Altman method,^8,12 concordance analysis, and inter-rater agreement Kappa. The discriminant cutoff values, sensitivity, and specificities of PCR were tested for predicting 24-hour protein by receiver operating characteristic (ROC) curves.¹³

Results

There was a positive correlation between spot urine (PCR) and 24-hour urine total protein (UP) (r = 0.7459, P < .0001; Figure 1). Wider deviation from the line of identity was seen at higher levels of protein excretion. Using the Bland-Altman plot, the limits of agreement between PCR and UP were +0.77 and -1.06 g/d. The limits of agreement of spot urine PCR and UP became wider at higher levels of protein excretion (Figure 2). Concordance analysis showed a concordance correlation coefficient of 0.6729 (95% confidence interval [CI]: 0.5344-0.7762; P < .0001). The Pearson ρ (precision) was 0.7459, and the bias correction factor Cb (accuracy) was 0.9021. The inter-rater agreement Kappa was calculated to be 0.451, indicating moderate agreement between the 2 methods.

Using the ROC analysis, the AUC for PCR at various cutoff levels was 0.967 (95% CI: 0.880-0.996; P < .0001; Figure 3). A sensitivity of 100% and specificity of 90% were achieved to detect proteinuria at a PCR greater than 0.433.

Discussion

The 24-hour urine protein excretion test is the criterion standard for quantitative protein analysis. An alternative to the 24-hour urine specimens is the urine PCR. A good correlation exists between the PCR and UP excretion, and has been demonstrated in patients with diabetic nephropathy,^11,14,15 renal transplant,^2,3,16 and pregnancy.¹⁷

The findings of this study also showed a good correlation between UP and PCR in untimed random specimens. To use 2 tests interchangeably, it is important to show that the 2 methods agree sufficiently. Few studies have assessed agreement rather than correlation between these tests, and have found wide limits of agreement.^9-11 In the present study, the limits of agreement were acceptable at low levels of protein excretion but became wide as the protein excretion increases. Concordance correlation coefficient was 0.6729 (95% CI: 0.5344-0.7762), Pearson ρ (precision) was 0.7459, and Bias correction factor Cb (accuracy) was 0.9021. The inter-rater agreement Kappa was calculated to be 0.451, indicating moderate agreement between the 2 methods.

Urinary protein excretion is not constant, and daily excretion varies by as much as 40%, and repeated 24-hour urine protein excretion by at least 15%.⁹ Rodby and associates repeated measurements on 33 patients and found discordant results: the PCR increased in some patients whereas the UP fell and vice versa.¹⁸ Agrawal found a day-to-day variability in 24-hour urinary protein excretion of 10% and in protein-to-creatinine ratio of 2%.¹⁹ This variability is a likely reason for the poor agreement between the 2 methods of assessing proteinuria.

Using ROC curve, the AUC for PCR at various cutoff levels was 0.967 (95% CI 0.880-0.996; P < .0001). A sensitivity of 100% and specificity of 90% were achieved to detect proteinuria at a PCR greater than 0.433. The sensitivity and specificity in the present study are consistent with previously published reports.^20-24 Price and associates²⁵ reviewed 16 studies, and the area under the curve was in the range of 0.70 to 0.90, suggesting that the PCR can predict the amount of protein excreted in the urine.

This study has limitations. First, the study has a relatively small numbers of subjects and includes more men than women. Second, the 2 samples of urine were collected within 2 days of each other, and this could affect the results significantly, as proteinuria may fluctuate from one day to another. We conclude that the PCR in spot urine specimens is an accurate, convenient, and reliable method of estimating the protein excretion in urine in patients who undergo a renal transplant, especially when proteinuria is at reasonably low levels.

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