enumerate the methods for estimation of urinary proteins and explain sulphosalicylic acid method by quantitative method
Answers
Answer:
The total proteins in human urine have been compared by sulfosalicylic acid, sulfosalicylic acid with sodium sulphate and trichloroacetic acid methods with pyrogallol red molybdate method as there are no studies found quantifying imprecision and bias components. Fresh urine of 36 patients was analyzed by four methods. Imprecision and inaccuracy were determined by repeated analysis and method comparison studies using correlation plots, Bland and Altman, and Passing and Bablok regression analyses respectively. The coefficient of variation was 5.07 % for pyrogallol red molybdate; 6.84 % for sulfosalicylic acid; 3.97 % for sulfosalicylic acid with sodium sulphate and 5.93 % for trichloroacetic acid methods. Bland and Altman analysis showed a bias of 5.8, 1.7 and −5.4 for pyrogallol red molybdate versus sulfosalicylic acid, sulfosalicylic acid with sodium sulphate and trichloroacetic acid methods respectively. Passing and Bablok regression revealed a constant bias for pyrogallol red molybdate versus all turbidimetric methods but a proportional bias only with trichloroacetic acid method. Sulfosalicylic acid with sodium sulphate method is preferred to sulfosalicylic acid and trichloroacetic acid methods.
Keywords: Urinary protein assays, Turbidimetric methods, Dye-binding methods, Correlation, Bland and Altman analysis, Passing and Bablok regression analysis
Introduction
The measurement of proteins in human urine provides an important tool in the diagnosis of renal diseases. Several methods are available for the estimation of total proteins in urine including turbidimetric methods such as sulfosalicylic acid (SSA) [1], sulfosalicylic acid with sodium sulphate (SSSS) [2, 3], trichloroacetic acid (TCA) [4], or benzethonium chloride (BEC) [5] and the protein dye-binding methods utilizing coomassie brilliant blue (CBB) [6, 7] or pyrogallol red molybdate (PRM) [8]. Among the turbidimetric methods Meulemans [3] found that SSSS method is better than SSA method and TCA method which has greater sensitivity and better reproducibility. Among the dye-binding methods PRM method is commonly used in most of the hospitals because it is more sensitive, precise and practicable [9].
The relative agreements between the different laboratory analytical methods that measure the same chemical substance are assessed by method comparison studies. There is paucity of Indian data on comparison of proteins in human urine by different methods [10]. Also there were no studies found showing the agreement between PRM and the turbidimetric methods in human urine samples. Hence in this study the results of total protein concentration in human urine obtained by SSA, SSSS and TCA methods were compared with the more commonly used PRM method to identify and quantify imprecision and bias components (both constant and proportional bias) using the advanced statistical techniques like Bland and Altman plots and Passing and Bablok regression.
Materials and Methods
Urine Samples
Fresh urine specimens of 36 patients collected from the central laboratory, MediCiti Institute of Medical Sciences (MIMS), Ghanpur, Ranga Reddy district, Telangana, India, between July and October 2014, without preservatives covering a wide range of protein concentrations (urine dipstick: nil, trace, 1+, 2+ and ≥3+) were randomly taken for analysis of total proteins. They were centrifuged (2500×g for 10 min) and then subjected to analysis by the four methods.
Reagents
Micro protein PRM kit and urine dip stick strips were purchased from Euro diagnostic systems, India Pvt. Ltd. Analytical grade reagents for SSA, SSSS and TCA methods were purchased from SD fine company.
Protein Assays
Urine Dipstick The dipstick test for total proteins is based on the “protein error of indicators” phenomenon in which certain chemical indicators demonstrate one colour in the presence of protein and another in its absence. The reagent is most sensitive to albumin and less sensitive to globulins, Bence–Jones protein, mucoproteins and haemoglobin. The test procedure was performed as described by the manufacturer. The colour was read exactly after 1 min. Colours formed range from yellow for a negative reaction to yellow green and green to blue green for a positive reaction [11].
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