Case report: Skin protective barrier cream interference in benzethonium chloride method for urine protein measurement in a 6-month-old girl

Introduction

Urine protein measurement is an essential diagnostic tool for monitoring renal function and disease. Among the numerous methods available for urine protein quantification, the benzethonium chloride (BTC) method has gained considerable attention due to its simplicity, rapidity, and cost-effectiveness. ¹ This colorimetric method is based on the formation of an insoluble complex between benzethonium chloride and urinary proteins, which can be measured spectrophotometrically at 630 nm. ² However, the accuracy of the BTC method can be affected by the presence of interfering substances (levodopa, methyldopa, Na2-cefoxitin, organically bound iodine from radiopaque media, high levels of homogentisic acid, and administration of gelatin-based plasma replacements) as is mentioned in the kit insert.^3–5

Urine collection bags are commonly used to get urine samples from babies. Barrier cream (BC) is a topical formulation which is frequently used to protect the sensitive skin of infants’ nappy area. This case report describes the positive interference of BC in the quantitative determination of urine protein by the BTC method in a 6-month-old girl suspected of having a congenital nephrotic syndrome (CNS). This case report for the first time highlights the impact of BC interference on the accuracy of urine protein measurements using the BTC method and emphasizes the importance of considering such interference when interpreting results.

Case presentation

A 6-month-old girl diagnosed with PMM2-CDG, a congenital disorder of glycosylation caused by phosphomannomutase 2 deficiency, was evaluated for renal abnormalities known to occur in this disease, especially nephrotic syndrome. ⁶ At 5 months old, her urine protein level was 0.7 g/L with a protein-to-creatinine ratio of 7730 mg/mmol creatinine (reference interval; <80 mg/mmol creatinine for 0.2–0.5 year ages), and her plasma albumin level was 30 g/L (reference interval; 25–46 g/L for 15 days–1 year age). At a specialized CDG centre a week later, her plasma albumin level decreased to 26 g/L and urine protein was not measured. The decreasing albumin level suggested she could be developing a nephrotic syndrome. At 6 months old, during a follow-up appointment with the nephrology department, a urine dipstick test was performed for the first time. Initial measurements using urine strips (Clinitek Status + Analyser, Siemens Healthcare Diagnostics) showed negative results for urine protein, while a quantitative determination using a commercial assay (Roche Diagnostics) based on the BTC method in a Roche Cobas 8000 c502 analyser (Roche Diagnostics, Hoffmann) revealed high protein levels (3.33 g/L, reference interval; <0.14). To evaluate this elevated protein concentration, immunometric urine albumin (Roche Diagnostics) and beta-2 microglobulin measurements (Roche Diagnostics) were performed. Although those levels were increased (119.41 mg/mmol creatinine (reference interval <3.5) and 0.25 mg/mmol creatinine (reference interval <0.030), respectively), these concentrations could not explain the high total protein concentration in urine. Furthermore, urine protein electrophoresis (Hydrasys 2 system, Sebia) showed no visible band for any protein type in the urine sample (Figure 1).OPEN IN VIEWER

Additionally, to investigate potential interference in the BTC method, the protein concentration in the patient urine sample was also measured by a pyrogallol red dye binding method (Beckman Coulter Inc.) on the Beckman Coulter DXC 700 AU analyser (Brea, CA USA) and found to be within the reference interval (0.08 g/L). These findings suggest a possible interference especially for the BTC method. To evaluate the size of interfering substance in the patient sample, urine samples from the patient and a positive control were subjected to ultrafiltration with a molecular weight cut-off of 10,000 Da (Millipore Amicon Ultra Centrifugal Filter Units 0.5 mL), resulting low protein levels in the ultrafiltrate (Table 1). This suggested the presence of a substance with high molecular weight causing positive interference.

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Table 1.

Protein levels in control and patient urine samples before and after the ultrafiltration with a 10,000 Da cut-off.

Sample	Protein (g/L) before ultrafiltration	Protein (g/L) after ultrafiltration
Positive control	12.2	0.28
Patient sample	1.66	0.02

Since urine samples from the patient obtained via catheter revealed a normal protein concentration (0.2 g/L), an external source for this interference was likely. We focused on the urine collection step for this baby. For babies, specific urine collection bags are needed to be placed on the genital area to get urine samples. Special skin protective creams are frequently used for protection of the sensitive skin in infants’ nappy areas. In our case, commercially available BC (Coloplast Conveen critic barrier cream) was used. As this cream contains some high molecular weight substances, it was suspected to be responsible for the positive interference. To simulate the urine and BC interactions during urine collection period, we incubated three urine samples (5 mL) together with smeared barrier cream on the bottom of a test tube and incubated for 120 min at 37°C, swirling occasionally. This procedure led to elevated protein levels (Table 2). Consequently, it was concluded that the initial extremely high protein levels measured using the BTC method were falsely elevated due to the interference of BC.

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Table 2.

Urine protein levels measured by the BTC method before and after mixing and incubation of urine sample with BC.

	Urine protein, g/L (BTC method)
Control urine (before incubation)	0.04
Control urine mixed with BC (1 h incubation)	0.89
Control urine mixed with BC (2 h incubation)	1.59

Discussion

High molecular weight substances that are present in barrier cream used together with urine collection bags may lead to positive interference in the benzethonium chloride method for urine protein measurements.

In this case, the initial extremely high protein levels obtained using the BTC method raised suspicion of interference, as they were inconsistent with the negative urine dipstick result. Due to the possibility of laboratory error, no further actions were taken regarding this high level of proteinuria. The medical team’s decision to repeat the urine protein measurement using the pyrogallol red method, which is less susceptible to interference, and the catheter urine showing no protein in the urine, helped to confirm the presence of interference and establish the correct protein levels in the patient’s urine.

This report presents the first case of positive interference by a barrier cream in urine protein measurements. The interference likely occurred due to the presence of high molecular weight molecules that may interfere with the BTC protein quantification method, leading to falsely elevated protein concentrations.

Conclusion

In patients with renal disease, it is crucial to ensure accurate measurements of urine protein levels. This case report highlights the importance of considering potential interferences, including those from barrier creams, when interpreting urine protein measurements. Clinicians and laboratory professionals should be aware of this potential interference and consider alternative collection methods, such as catheterization, or measurement with dye binding methods to obtain accurate results in patients using such products. Further research is warranted to investigate the extent of interference caused by high molecular weight substance-containing products and develop strategies to mitigate their impact on clinical measurements.