Urine specimens
284 urine specimens were randomly selected among SLE patients from the department of rheumatology and immunology in Quanzhou First Hospital between January 2021 and December 2021. The patients were females whose ages ranged from 13 yrs to 73yrs with the median age being 36 years. The protocol was approved by the Ethics Committee of Quanzhou First Hospital (China), and the protocol number is [2021] 193. According to the incidence of renal injury complicated, they were divided into the SLE group (163 females, aged 13–66 years, median age 34 years) and the LN group (124 females, aged 20–73 years, median age 37 years). Meanwhile, healthy women were selected as the control group (336 females, aged 21–69 years, median age 37 years).
Inclusion standards
SLE group: (1) Patients who were with a final diagnosis of SLE made by the clinician in accordance with the 2012 systemic lupus erythematosus international lupus research clinical collaboration group (SLICC) or SLE classification standard formulated by EULAR / ACR in 2019[12]. (2) Patients were excluded when they were with diabetes, allergic purpura and other diseases that may cause acute and chronic kidney injury, or patients with kidney disease and other urinary system diseases. (3)Proteinuria stably lower than or equal to the level of 500 mg/24 h, or random proteinuria < 3+, or P/C ≤ 500 mg/g(50 mg/mmol). (4) Microscopic examination of urine sediment showed no cell cast (erythrocyte cast, haemoglobin cast, granular cast, tubular cast, and mixed cast) or no active urine sediment (white blood cells excluding ≤ 5/HP, red blood cells ≤ 5/HP, excluding urinary tract infection).
LN group: (1) Clinically diagnosed as SLE, (2) Patients were excluded when they were with acute and chronic kidney injury caused by diabetes, allergic purpura, or with nephropathy and other urinary system diseases. (3) Proteinuria stably overcame the level of 500 mg/24 h, or random proteinuria 3+, or P/C ratio > 500 mg/g(50 mg/mmol)[7]. (4) Microscopic examination of urine sediment showed several kinds of cell cast (erythrocyte cast, haemoglobin, granular cast, tubular cast, and mixed cast), or active urine sediment (white blood cells excluding > 5/HP, red blood cells > 5/HP, excluding urinary tract infection).
Control group: (1) Normal renal function and urine test. (2) Normal imaging examination and physical examination. (3) No acute and chronic kidney injury caused by diabetes, allergic purpura, or patients with nephropathy and other urinary system diseases. (4) No history of systemic diseases such as hepatitis, rheumatism, and urinary system diseases.5) No history of major surgery, 6) Not taking drugs in recent one month, 7) Non-menstrual.
Sample detection
Mid-stream specimens were collected in sterile containers and were tested within two hours. Each specimen was divided into two sterile tubes, with 10ml urine being tested by Beckman AU5800 automatic biochemical analyzer and the other tube of 10ml urine by UN2000. Urine protein, creatinine, P/C, and RTEC were detected using both analyzers.
Conventional microscopic examination
8ml of urine sample was centrifuged for 5 min at 400 g (1500 rpm) before the supernatant fluid was taken out and about 100ul sediment was left. After mixing the sediment, 1 drop of S-M (sternheirnermalbin) staining solution was added for one-minute staining. After that, approximately 20µl was put onto a microscope slide which was then examined under a CX23 microscope (Olympus, Tokyo, Japan) by 2 medium-grade professionals titled clinical laboratory technicians. The average value of the two measurements for RTEC was calculated. The two technicians had completed the personnel comparison; the coincidence rate ≥ of 80% represented passing.
Automated urine sediment analyzers and reagents
UN2000 was provided by Sysmex Corporation, Japan. AU5800 was provided by Beckman Coulter Corporation, American. Sysmex Corporation provided all reagents, quality control material, and calibrator. All of them were used within the validity period. Regular calibration and performance verification had been previously performed. The urine was detected everyday after passing the quality control whose result conformed to ISO 15189. TD4N centrifuge (low-speed urine centrifuge) was provided by Shanghai Luxiangyi centrifuge corporation. S-M staining solution was purchased from Baso Corporation, Zhuhai, China. CX23 binocular microscope was supplied by Japan Olympus Corporation, Japan.
Statistical analysis
Accuracy evaluation of P/C
The P/C results detected by AU5800 were used as the gold standard. According to the UN2000 test strip instruction book, the instrument result is negative when P/C < 150 mg/g, which is positive (+) when P/C (150–499) mg/g and positive (2+) when P/C ≥ 500 mg/g. Kappa’s concordance coefficient was calculated for the agreement between the instruments. Kappa values were considered to be poor agreement (0 ~ 0.20), fair (0.21 ~ 0.40), moderate agreement (0.41 ~ 0.60), good agreement (0.61 ~ 0.80) and excellent agreement (0.81 ~ 1.00).
Accuracy evaluation of RTEC
Establishment of reference intervals and verification of RTEC.
RTEC results were extracted from the control group of 316 healthy women to establish the reference. The detection data were non-normally distributed. Consequently, referring to the CLSI C28-A3 document [13], (0-P95) was set as the reference range after excluding outliers in the data while 20 healthy women were selected from the control group to verify the reference interval. 90% of the test results passed the verification within the established reference range.
With the conventional microscopic results as the golden standard, positive was defined when RTEC was found by conventional microscopic examination, while it was negative when no RTEC was detected. When the results of UN2000 were greater than the reference range established in this study, the results were positive. Otherwise, the results were negative [11]. The negative and positive coincidence rates were analyzed by Kappa consistency test.
Comparison of RTEC levels in the control group, SLE group, LN group, and ROC curve analysis
SPSS21.0 was used for statistical analysis. The results of RTEC were expressed by mean and standard deviation. Kruskal Wallis test was used for comparison among groups. Mann Whitney U test was used for comparison between groups. The receiver operating characteristic (ROC) curve was used to analyze the ability of RTEC to screen LN. A further analysis was performed on the diagnostic efficacy of single item or combination of P/C and RTEC for screening LN.