DOI 10.1515/cclm-2013-0868 Clin Chem Lab Med 2014; 52(4): e71–e73
Letter to the Editor Hatice Sürer, Tuba Özgün, Fatma Meriç Yilmaz and Gülsen Yilmaz*
The effect of centrifugation on three urine protein assays: benzethonium chloride, benzalkonium chloride and pyrogallol red Keywords: benzethonium chloride; benzalkonium chloride; centrifugation; pyrogallol red; urine total protein. *Corresponding author: Gülsen Yilmaz, S.B. Ankara Eğitim ve Araştırma Hastanesi, Tıbbi Biyokimya Bölümü, 06340 Mamak/ Ankara, Turkey, Phone: +90 312 5953218, Fax: +90 312 3621857, E-mail: [email protected] Hatice Sürer, Tuba Özgün and Fatma Meriç Yilmaz: Department of Medical Biochemistry, Ankara Numune Training and Research Hospital, Ministry of Health, Ankara, Turkey
To the Editor,
Laboratory measurement of urine total protein has been important for the diagnosis and monitoring of renal diseases for decades. Pyrogallol red (PYR) and benzethonium chloride (BTC) have become widely used automated urine protein assays in the clinical laboratory. It is recommended that samples should ideally be analysed fresh and urine should be centrifuged prior to analysis [1]. However, in routine practice, we observed that centrifugation can be neglected as an additional preanalytical step. The present study was carried out to investigate the impact of centrifugation on protein levels by analysing urine samples with normal and elevated protein concentration and the relationship between urinary tract infection, a common cause of cloudy urine, and the percentage change in protein levels after centrifugation. The possible effect of centrifugation on urine protein levels was investigated for benzalkonium chloride (BC), BTC and PYR assays. We previously reported a turbidimetric BC assay as an alternative automated urinary protein method and indicated the effect of hemolysis and secondary wavelength on our method. We have been using automated BC method with in-house reagents in our routine biochemistry laboratory [2–4]. In order to investigate the possible effect of centrifugation on protein levels, random midstream urine
specimens (n = 112) were obtained from patients for routine urine analysis. Exclusion criteria included menstrual cycle at the time of collection and urine samples with mucus that could be seen visually. The study was approved by the Institutional Ethical Committee. Before protein and creatinine measurements, urine dipstick tests [leukocyte esterase (LE)] were performed for every specimen. Protein and creatinine measurements were carried out before and after centrifugation (1000 g for 5 min). Protein levels were measured using three different methods: BC, BTC and PYR. BC urine protein, PYR urine protein and creatinine levels were determined in an Olympus 640 analyser. Inhouse reagents for BC and commercial reagents (Mishima Olympus Co Ltd., Olympus Corporation, Japan) for PYR and creatinine assays were used. BTC protein levels were determined on a Modular P analyzer with commercial reagents (Roche Diagnostics, Mannheim, Germany). As urine protein values under 140 mg/L were accepted as normal, two groups were composed according to their protein concentration. Group A: Urinary protein 140 mg/L (n = 59). The data was further subdivided into LE-negative and -positive subgroups. All statistical analyses were performed using SPSS software (version 15.0). Distribution of the data was evaluated by a Kolmogorov-Smirnov test. The protein levels before and after centrifugation were compared by using paired t-test. The change in urinary protein levels by centrifugation in dipstick test positive and negative groups were determined using a Mann-Whitney test. p-Values 0.05). Dye-binding PYR and turbidimetric BTC methods for the determination of total urinary protein are rapid, simple, and widely used automated urine protein assays in the clinical laboratory. Urine protein concentrations
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Sürer et al.: The effect of centrifugation on three urine protein assays: benzethonium chloride e73
before centrifugation were statistically significantly higher than the urinary protein concentrations after centrifugation for not only BTC and PYR but also BC. BC and BTC methods which are based on turbidimetric measurements, showed higher percentage changes than the PYR method. It can be concluded that the effect of centrifugation on urine protein concentrations is observed especially in low protein concentrations and the magnitude of the change differs according to the method used to determine the urine protein. Turbidimetric methods seem to be more affected than PYR method, and this can be due to the turbidity in urine samples which is eliminated with centrifugation. The percentage changes in group B were much lower of those observed in group A indicating that the effect of centrifugation might be more important in low protein concentrations. Although PCRs have been widely used in clinical practice, we compared our data according to protein concentrations to underline the effect of different protein assays in analytical aspects. As urine protein values under 140 mg/L was accepted as normal [1], determination of urine protein without centrifugation
near cut-off values can cause false-positive results in clinical practice. Urine is a complex fluid with a large variation in content, and it is difficult to determine the specific components of urine that may interfere with the assay. In conclusion, false-positive urine protein results can impact diagnosis and patient care. The present study is a preliminary study that highlights the need for laboratory quality improvement projects and standardisation of laboratory urine protein measurement with preanalytical aspects. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received October 10, 2013; accepted October 30, 2013; previously published online November 27, 2013
References 1. Wu AH, editor. Tietz clinical guide to laboratory tests, 4th ed. St. Louis: Elsevier Saunders, 2006:916. 2. Yilmaz FM, Celebi N, Yücel D. Automated turbidimetric benzalkonium chloride method for measurement of protein in urine and cerebrospinal fluid. Clin Chem 2004;50:1450–2. 3. Yilmaz FM, Yücel D. Effect of addition of hemolysate on urine and cerebrospinal fluid assays for protein. Clin Chem 2006;52:152–3. 4. Yilmaz FM, Yilmaz G, Yücel D. Is secondary wavelength always necessary in turbidimetric urine protein measurements? Clin Biochem 2008;41:645–7. 5. Lamb EJ, Price CP. Kidney function tests. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical
chemistry and molecular diagnostics. St. Louis: Elsevier Saunders, 2012:669–708. 6. Saetun P, Semangoen T, Thongboonkerd V. Characterizations of urinary sediments precipitated after freezing and their effects on urinary protein and chemical analyses. Am J Physiol Renal Physiol 2009;296:F1346–54. 7. Johnson DW. Global proteinuria guidelines: are we nearly there yet? Clin Biochem Rev 2011;32:89–95. 8. Price CP, Newall RG, Boyd JC. Use of protein:creatinine ratio measurements on random urine samples for prediction of significant proteinuria: a systematic review. Clin Chem 2005;51:1577–86.
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Letter to the Editor Hatice Sürer, Tuba Özgün, Fatma Meriç Yilmaz and Gülsen Yilmaz*
The effect of centrifugation on three urine protein assays: benzethonium chloride, benzalkonium chloride and pyrogallol red Keywords: benzethonium chloride; benzalkonium chloride; centrifugation; pyrogallol red; urine total protein. *Corresponding author: Gülsen Yilmaz, S.B. Ankara Eğitim ve Araştırma Hastanesi, Tıbbi Biyokimya Bölümü, 06340 Mamak/ Ankara, Turkey, Phone: +90 312 5953218, Fax: +90 312 3621857, E-mail: [email protected] Hatice Sürer, Tuba Özgün and Fatma Meriç Yilmaz: Department of Medical Biochemistry, Ankara Numune Training and Research Hospital, Ministry of Health, Ankara, Turkey
To the Editor,
Laboratory measurement of urine total protein has been important for the diagnosis and monitoring of renal diseases for decades. Pyrogallol red (PYR) and benzethonium chloride (BTC) have become widely used automated urine protein assays in the clinical laboratory. It is recommended that samples should ideally be analysed fresh and urine should be centrifuged prior to analysis [1]. However, in routine practice, we observed that centrifugation can be neglected as an additional preanalytical step. The present study was carried out to investigate the impact of centrifugation on protein levels by analysing urine samples with normal and elevated protein concentration and the relationship between urinary tract infection, a common cause of cloudy urine, and the percentage change in protein levels after centrifugation. The possible effect of centrifugation on urine protein levels was investigated for benzalkonium chloride (BC), BTC and PYR assays. We previously reported a turbidimetric BC assay as an alternative automated urinary protein method and indicated the effect of hemolysis and secondary wavelength on our method. We have been using automated BC method with in-house reagents in our routine biochemistry laboratory [2–4]. In order to investigate the possible effect of centrifugation on protein levels, random midstream urine
specimens (n = 112) were obtained from patients for routine urine analysis. Exclusion criteria included menstrual cycle at the time of collection and urine samples with mucus that could be seen visually. The study was approved by the Institutional Ethical Committee. Before protein and creatinine measurements, urine dipstick tests [leukocyte esterase (LE)] were performed for every specimen. Protein and creatinine measurements were carried out before and after centrifugation (1000 g for 5 min). Protein levels were measured using three different methods: BC, BTC and PYR. BC urine protein, PYR urine protein and creatinine levels were determined in an Olympus 640 analyser. Inhouse reagents for BC and commercial reagents (Mishima Olympus Co Ltd., Olympus Corporation, Japan) for PYR and creatinine assays were used. BTC protein levels were determined on a Modular P analyzer with commercial reagents (Roche Diagnostics, Mannheim, Germany). As urine protein values under 140 mg/L were accepted as normal, two groups were composed according to their protein concentration. Group A: Urinary protein 140 mg/L (n = 59). The data was further subdivided into LE-negative and -positive subgroups. All statistical analyses were performed using SPSS software (version 15.0). Distribution of the data was evaluated by a Kolmogorov-Smirnov test. The protein levels before and after centrifugation were compared by using paired t-test. The change in urinary protein levels by centrifugation in dipstick test positive and negative groups were determined using a Mann-Whitney test. p-Values 0.05). Dye-binding PYR and turbidimetric BTC methods for the determination of total urinary protein are rapid, simple, and widely used automated urine protein assays in the clinical laboratory. Urine protein concentrations
Brought to you by | National Chung Hsing University Authenticated | 140.120.135.222 Download Date | 3/30/14 12:39 AM
Sürer et al.: The effect of centrifugation on three urine protein assays: benzethonium chloride e73
before centrifugation were statistically significantly higher than the urinary protein concentrations after centrifugation for not only BTC and PYR but also BC. BC and BTC methods which are based on turbidimetric measurements, showed higher percentage changes than the PYR method. It can be concluded that the effect of centrifugation on urine protein concentrations is observed especially in low protein concentrations and the magnitude of the change differs according to the method used to determine the urine protein. Turbidimetric methods seem to be more affected than PYR method, and this can be due to the turbidity in urine samples which is eliminated with centrifugation. The percentage changes in group B were much lower of those observed in group A indicating that the effect of centrifugation might be more important in low protein concentrations. Although PCRs have been widely used in clinical practice, we compared our data according to protein concentrations to underline the effect of different protein assays in analytical aspects. As urine protein values under 140 mg/L was accepted as normal [1], determination of urine protein without centrifugation
near cut-off values can cause false-positive results in clinical practice. Urine is a complex fluid with a large variation in content, and it is difficult to determine the specific components of urine that may interfere with the assay. In conclusion, false-positive urine protein results can impact diagnosis and patient care. The present study is a preliminary study that highlights the need for laboratory quality improvement projects and standardisation of laboratory urine protein measurement with preanalytical aspects. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received October 10, 2013; accepted October 30, 2013; previously published online November 27, 2013
References 1. Wu AH, editor. Tietz clinical guide to laboratory tests, 4th ed. St. Louis: Elsevier Saunders, 2006:916. 2. Yilmaz FM, Celebi N, Yücel D. Automated turbidimetric benzalkonium chloride method for measurement of protein in urine and cerebrospinal fluid. Clin Chem 2004;50:1450–2. 3. Yilmaz FM, Yücel D. Effect of addition of hemolysate on urine and cerebrospinal fluid assays for protein. Clin Chem 2006;52:152–3. 4. Yilmaz FM, Yilmaz G, Yücel D. Is secondary wavelength always necessary in turbidimetric urine protein measurements? Clin Biochem 2008;41:645–7. 5. Lamb EJ, Price CP. Kidney function tests. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical
chemistry and molecular diagnostics. St. Louis: Elsevier Saunders, 2012:669–708. 6. Saetun P, Semangoen T, Thongboonkerd V. Characterizations of urinary sediments precipitated after freezing and their effects on urinary protein and chemical analyses. Am J Physiol Renal Physiol 2009;296:F1346–54. 7. Johnson DW. Global proteinuria guidelines: are we nearly there yet? Clin Biochem Rev 2011;32:89–95. 8. Price CP, Newall RG, Boyd JC. Use of protein:creatinine ratio measurements on random urine samples for prediction of significant proteinuria: a systematic review. Clin Chem 2005;51:1577–86.
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