Designing and evaluating analytical parameters to adapt siemens urinary creatinine enzymatic method to open system analysers

Urinary creatinine is measured to assess kidney function and also as part of sample validity testing in drugs of abuse. Creatinine methods based on alkaline picrate Jaffé’s reaction require extra cleaning on chemistry analysers to minimise any interference from picric acid and sodium hydroxide on other reagents on board. Enzymatic methods reagents are not as invasive and more specifi c. Siemens enzymatic method is more sensitive and specifi c when compared to Thermo Fisher alkaline picrate method but there were no available analytical parameters to setup the Siemens enzymatic method on Beckman-Coulter AU5800 analyser as an open system analyser. Emulating setup parameters from Siemens chemistry analysers did not work. The analytical parameters were developed through systematic testing using different settings to adopt and optimise the method. A full correlation was performed using the developed parameters with alkaline picrate method. The Deming regression weighted analysis for 438 samples showed a good correlation at a 95% confi dence interval. Slope is 1.029 to 1.041, Y-intercept when X=0.0 is -0.9156 to -0.7481 and correlation coeffi cient (r) is 0.998. Alkaline picrate method Mean and SD is 12.059 and 7.248 respectively and for the Enzymatic method is 11.653 and 7.504 respectively. Many interferences from drugs and other substances are eliminated when using the enzymatic method and the stability of other reagents on-board improved because the reagents used in the enzymatic method are less invasive. Procedures Designing and evaluating analytical parameters to adapt siemens urinary creatinine enzymatic method to open system analysers Ashraf Mina1,2*, Leah McNeice1, Shanmugam Banukumar1 and Santiago Vazquez1 1NSW Health Pathology, Forensic & Analytical Science Services (FASS), Toxicology Unit, Macquarie Hospital, NSW, Australia 2Affi liated Senior Clinical Lecturer, Faculty of Medicine and Health, Sydney University, NSW, Australia Received: 04 September, 2020 Accepted: 16 September, 2020 Published: 17 September, 2020 *Corresponding author: Dr. Ashraf Mina, NSW Health Pathology, Forensic & Analytical Science Services (FASS), Toxicology Unit, Macquarie Hospital, NSW, Australia, Tel: +61 02 9887 5666, +61 0403 108 682; Fax: +61 02 9805 1259; E-mail:


Introduction
Creatinine is a chemical waste product that is produced from normal wear and tear on muscles metabolism and to a smaller extent by eating meat. Healthy kidneys fi lter creatinine and other waste products from your blood. These waste products are removed from the body through urination. Urinary Creatinine is used to assess the kidney function by measuring the glomerular fi ltration rate (GFR). Also, urinary creatinine is analysed as part of urine sample validity when testing for drugs of abuse. Urine creatinine is used as an indicator of urine water content and as a sample validity test for urine specimens.
Large intake of fl uids will increase the urine water content and decrease the creatinine level and as a result diluting the drug concentration in urine. Creatinine levels lower than reference intervals in drug testing indicate that a person has been drinking a lot of fl uids and attempted to dilute the results to cover the consumed drug and its metabolites.
Citation: Mina  The creatinine Jaffé's method (alkaline picrate method) is prone to bias because of interfering substances, which means less analytical specifi city. Additionally, the Jaffé's method may represent a low risk in some patients if the estimated Glomerular Filtration Rate (eGFR) result is around the 60 ml/min/1.73 m 2 decision limit, which in that case should be interpreted with caution [1,2].
In patients on dialysis, when creatinine was assayed in peritoneal dialysis solutions and pure glucose solutions, the Jaffé's reaction overestimated the results due to other components of dialysis solutions, mainly calcium chloride.
The specifi c enzymatic method is a more accurate, specifi c and reliable assay for creatinine kinetics through the peritoneal membrane when determined in Continuous Ambulatory Peritoneal Dialysis (CAPD) solutions [3].
Albumin, Immunoglobulin G (IgG), and Hemoglobin F (Hb F) do not interfere with the enzymatic methods but they interfere with Jaffé's creatinine assays, leading to inaccuracies in eGFR that are clinically important, especially in neonates and children. Consequently, the enzymatic creatinine methods are preferred for evaluation of kidney function in pediatric patients [4]. Although Jaffé and enzymatic methods meet the analytical performance requirements in routine use, the enzymatic method performed better in measuring low creatinine levels [5]. For the above-mentioned reasons, using enzymatic creatinine as part of sample validity testing for drugs of abuse will give more reliable results.
Analytically, the enzymatic method has many advantages over Jaffé-based methods such as smaller sample size, faster sample throughput, and more specifi city. Additionally, glucose, acetoacetate, and cefoxitin don't interfere with the enzymatic method, but bilirubin may cause a negative interference depending on both creatinine and bilirubin concentrations.
Clinically, the enzymatic method is preferred in monitoring, neonates, diabetic ketotic patients, renal disorders patients and those receiving cephalosporins [6,7]. Some cephalosporinlike antibiotics interfere with Jaffé's methods [8]. Siemens enzymatic method states that patients undergoing treatment with Phenindione and Dobesilate may show falsely low results.
Cefoxitin showed -11% bias in serum creatinine at 2230 μg/mL Many studies showed that the enzymatic creatinine method performed better than was better than methods based on Jaffé's reaction in terms of specifi city and sensitivity [9][10][11][12][13].

Materials and methods
The reagents used for this evaluation and their part Program is used as another tool to verify the outcome. The developed parameters to adopt the Siemens enzymatic creatinine method for urinary creatinine are summarised in

Results
A correlation and regression analysis (specimen equivalency) between Thermo Fisher creatinine alkaline picrate method and Siemens creatinine enzymatic method were performed using weighted Deming regression analysis. The number of patients' samples analysed is 438. Figure 1 and Table 2 summarise the data analysis.
Citation: Mina  Correlation coeffi cient (r) is 0.998. If the (r) value is greater than 0.7, the correlation is considered strong. If between 0.5 and 0.7, it is considered a moderate correlation. If (r) value is less than 0.4, the correlation is considered weak. The calibration curve is shown in Figure 2.
The Siemens creatinine enzymatic method was accepted according to our laboratory acceptance criteria which are no big difference in SD, the slope is 1.0 or close to one and correlation coeffi cient (r) is more than 0.7.
The Limit of Detection (LoD) corresponds to the lowest concentration of creatinine that can be detected with a probability of 95%. LoD is ≤ 1.0 mg/dL (0.0884 mmol/L) for urine. The measuring interval for urine is 1.00 -245.00 mg/dL (0.0884 -21.66 mmol/L). The extended measuring interval was 3674.93 mg/dL (324.87 mmol/L). The developed Parameters is set up for the extended measuring interval to avoid multiple sample dilutions beyond the measuring interval. The imprecision of the creatinine assay for within run and between runs is ≤ 5.0% Total CV.
The results of the samples tested from a national External Quality Assurance Scheme (EQAS) Austox Urine Profi ciency Program is summarised below in Table 3.
Measurement Uncertainty (MU) was calculated at different levels by multiplying each assay Standard Deviation (SD) by 1.96 to cover 95% confi dence interval of the tested samples based on the recommended model of the Royal College of Pathologists of Australasia (RCPA). Table 3 shows urinary creatinine Measurement Uncertainty (MU) at four different Levels in mmol/L Table 4.

Discussion
When it comes to either assessing the renal function or sample validity testing in urine, laboratories must determine the urinary creatinine level. Normal creatinine level indicates the urine sample is undiluted, but low creatinine concentration indicates the urine sample has either been adulterated or manipulated.
Urinary creatinine clearance in urine and consequently GFR determined by Jaffé's method was less than that obtained by the enzymatic method when the serum creatinine concentration is less than 2.0 mg/dl [15]. Creatinine should be reported as "dilute" when the creatinine concentrations are equal to or more than 0.1768 mmol/L (2 mg/dL) and less than 1.7680 mmol/L (20 mg/dL).
Laboratories testing for drugs of abuse should use creatinine concentration as a trigger to conduct further validity testing.
If the creatinine level is less than 1.7680 mmol/L (20 mg/dL), then the laboratory should also measure specifi c gravity level which should be more than 1.0010 but less than 1.0030 and pH is between the mmol/L intervals 4.   Alkaline picrate method SD for tested samples 7.248 Enzymatic method SD for tested samples 7.504 Citation: Mina  If a dilute specimen tested positive for a drug, it should be reported as "positive dilute specimen" and is considered positive. If a dilute specimen tested negative for a drug, it should be reported as "negative dilute specimen" and is considered negative. The fact that it is a dilute specimen is irrelevant.
One study claimed that the level of interference from dextrose with enzymatic assay was greater at a higher creatinine concentration and concluded that the enzymatic assay may not be appropriate for patients using dialysate with dextrose 4.25% [16]. This study failed to mention which enzymatic method was used. The glucose reference interval in urine is 0 to 0. Another study evaluated the Kodak Ektachem analyser dry-slide creatinine enzymatic method using creatinine iminohydrolase in serum found that the method had no interference from substances that interfere with Jaffé's methods such as acetoacetate, cephalothin and cephoxitin, but 5-fl uorocytosine interfered signifi cantly. The study concluded that the enzymatic method fast, specifi c and precision and, except for one drug when compared to Jafee's method [17].
While alkaline picrate and enzymatic creatinine methods have good precision, the enzymatic method is more precise and able to detect a biologic change in creatinine faster and consequently can detect earlier clinically signifi cant changes when assessing renal function [18].
From practical experience, we found that due to the yellow colour of picric acid reagent used in the Jaffé's method, the cleaning of the reaction cuvettes has to be comprehensive to avoid contaminating other assays on-board. Also, the sodium hydroxide used in Jaffé's method can affect the stability of other reagents. Some drugs, such as cyclosporine were affected and its stability was reduced to only one day when the assay reagents were placed nearby Jaffé's method reagents on the analyser reagents carousels.