Research article · 2020 · Full text
International Journal of Advanced Biochemistry ResearchComparison of 150mmol/L and 250mmol/L concentration of sodium hydroxide in Jaffe's two point method for serum creatinine measurement with respect to linearity of assay
Volume 4(1) · Pages 42–46 · DOI: 10.33545/26174693.2020.v4.i1a.79 ↗
Research areasClinical Laboratory Practices and Quality ControlHemodynamic Monitoring and Therapy
Abstract
Author-writtenObjectives: Objective of the study is to perform linearity check for Creatinine measurement with Jaffe's two point method using CLSI EP-6 guideline for 150 mmol/L, 250mmol/L NaOH concentration in final reagent mixture. Material & Method: Leftover serum of samples received by the laboratory, devoid of patient identification, was used to make pool of serum. Serum creatinine reagents have NaOH of 150 mmol/L, 250 mmol/L were prepared. Serial dilution of pooled serum spiked with creatinine powder was prepared. Creatinines were measured in series of dilution. Linear regression analysis of each dilution series to find linearity limit was performed. esult & Discussion: This study evaluated Linearity difference plot using CLIA criteria (15% or 0.3 mg/dl whichever is higher) for creatinine at 95% confidence limit.7.5% of result or 0.15 mg/dl were used for plotting Linearity Difference plot. If any the two conditions are satisfied at a given point in the Linearity Difference Plot, the point is considered within linearity. 250 mmol/L NaOH in final reagent mixture gives limit of linearity 23.77mg/dl and 150 mmol/L NaOH in final reagent mixture gives limit of linearity 31.16 mg/dl. Conclusion: 150 mmol/L final reagent concentration of NaOH gives higher linearity of 31.16 mg/dl, low slop (a=29.23) indicating high sensitivity of the reagent, low y-intercept (b=-0.4578) indicating minimum effect of non-creatinine chromogens; i.e. high specificity than 250mmol/L final reagent concentration of NaOH.
Summary
Source-checkedPlain-language reading of the full paper, prepared with AI and checked against the text. Each point links to the authors' own words.
Why it matters
- Serum creatinine measurement is a common clinical chemistry test used to diagnose, monitor treatment, and judge the prognosis of renal disorders. View evidence ↗
- For clinical utility, creatinine reports must be accurate and precise, requiring well-defined parameters such as the limit of linearity. View evidence ↗
- A higher limit of linearity (LoL) is beneficial because it reduces the need for sample dilution and repeat testing, thereby lowering costs. View evidence ↗
The question
- The study aimed to compare the limit of linearity (LoL) of Jaffe's reaction mixture using two different sodium hydroxide (NaOH) concentrations: 150 mmol/L and 250 mmol/L. View evidence ↗
What they did
- The researchers used a pool of de-identified leftover serum samples from the laboratory. View evidence ↗
- A high-concentration serum pool was created by dissolving 25 mg of creatinine powder in 25 ml of the serum pool, while the remaining serum served as the low pool. View evidence ↗
- Following CLSI EP-6A guidelines, an 11-point serial dilution was performed using the high and low serum pools. View evidence ↗
- Measurements were conducted in four replicates for each dilution using an Erba XL 640 autoanalyser. View evidence ↗
- The linearity difference plots were based on CLIA acceptability criteria, using limits of 7.5% of the result or 0.15 mg/dl. View evidence ↗
What they found
- The 150 mmol/L NaOH reagent demonstrated a higher limit of linearity (31.16 mg/dl) compared to the 250 mmol/L NaOH reagent (23.77 mg/dl). View evidence ↗
- The 150 mmol/L NaOH reagent had a slope of 29.23 and a y-intercept of -0.4578, while the 250 mmol/L reagent had a slope of 28.27 and a y-intercept of -0.6996. View evidence ↗
- The 150 mmol/L reagent showed higher sensitivity due to its slope and lower interference from non-creatinine chromogens due to its lower y-intercept. View evidence ↗
What it means
- The authors concluded that the 150 mmol/L NaOH concentration is superior to the 250 mmol/L concentration because it provides higher linearity, higher sensitivity, and higher specificity. View evidence ↗
Visual summary
2 visualsIllustrations explain the paper; charts are replotted from the paper's own table values.
Comparison of Linearity Limits
AI-generated conceptual illustration (glass test tubes containing yellow-orange chemical reagents and serum). It explains the idea and does not show study data.
View source evidence ↗
Limit of Linearity (LOL) (mg/dl): 150 vs 250
Replotted from Table 1 (page 5) of the original paper. Every bar is the paper's own table value.
View source evidence ↗Figures & tables
From the original paper
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Table 1
Figure 9Introduction
Creatinine measurement in serum is one of the most frequently used laboratory examination in clinical chemistry laboratory. It is used in diagnosis, treatment monitoring and judging prognosis of renal disorders. In order to be useful to the clinicians, the creatinine reporting should be accurate, precise, free from interference. It should have well defined limit of blank, limit of detection, limit of qualification and limit of linearity. It is required to compare NaOH concentration of these reactants to improve its clinical usefulness. This study aims at comparing 150mmol/L and 250mmol/L NaOH concentration in Jaffe's reaction mixture with respect to limit of linearity (LOL). Measurement of Limit of Linearity is essential to device alternate strategy for measuring samples when results are beyond the limit. Measurement of Limit of Linearity is essential to device alternate strategy for measuring samples when results are beyond the limit.
Materials and Methods
Leftover serum of samples received by the laboratory, devoid of patient identification, was used to make pool of serum which had creatinine concentration less than 1.0mg/dl with no visible lipemia, hemolysis and icterus. Higher concentration pool was made by dissolving 25mg creatinine powder in 25ml leftover serum pool. Remaining serum pool was used as low serum pool. Serum creatinine reagent 1 were made by adding SDS 3.47mmol/L and Brij 3gm/L and NaOH 300mmol/L in which final reagent mixture have NaOH reagent mixture have NaOH of 150 mmol/L. Serum creatinine reagent 1 were made by adding SDS 3.47mmol/L and Brij 3gm/L and NaOH 500mmol/L in which final reagent mixture have NaOH reagent mixture have NaOH of 250 mmol/L. Creatinine reagent 2 were made by adding picric acid 20mmol/L and Brij 3gm/L. 11 point serial dilution of prepared higher pool with lower pool was done as recommended by CLSI EP-6A guideline [1] . Calibration of each reagent was done by Randox Normal Calibrator (828UN), Randox Abnormal Calibrator (591UE) and Bovine Albumin Blank (5 gm%) whose assigned value was 1.37mg/dl, 4.01mg/dl and 0.0 mg/dl respectively.
Creatinine measurement with each reagent in each member of series of dilution in 4 replicates were performed in Erba XL 640 autoanalyser. Graph of relative concentration vs. observed concentration was plotted in. First, Second and third order polynominals were found for each plot using spreadsheet software. Deviations from linearity (DLi) were evaluated as follows for each point of the plot. DL2i were used to analyse linearity as it was closed to 1. CLIA acceptability criteria was used to analyse the sample.
Results
Following tables and graphs shows results of the study. Above graph shows relationship between relative concentration and obtained concentration. Relative concentration is taken from 0 to 100; as per CLSI EP-6A, any values can be used as relative concentrations so far as their relative values remain same [1] .
Results of
Creatinine Linearity Difference Plot for 150 mmol/L NaOH concentration, Stage-1: As described in CLSI EP-6A, calculations were done for making Linearity Difference plot. The plot requires definition of acceptability criteria. This study evaluated Linearity difference plot using CLIA criteria for creatinine at 95% confidence limit. As CLIA acceptability for creatinine is 15% or 0.3 mg/dl whichever is higher, 7.5% of result or 0.15 mg/dl were used for plotting Linearity Difference plot. If any the two conditions are satisfied at a given point in the Linearity Difference Plot, the point is considered within linearity. Stage-1 is Linearity Difference Plot which includes all the 11 points. Stage-2 is Linerarity Difference Plot which includes only those points which make linearity evaluation acceptable at all points. The calculations of Linearity Difference plot are explained in table 11 for 150 mmol/L NaOH. Above graph shows that except point 1, all other points are within the ±7.5% limit. By both, % and mg/dl criteria, point 1 is unacceptable, so, the analysis is repeated by removing highest point, one by one till the all the points come within the acceptable criteria. Above graph shows that except point 1, all other points are within the ±7.5% limit.Only 1 point is within the 0.15mg/dl. By both, % and mg/dl criteria, point 1 is unacceptable, so, the analysis is repeated by removing higest point, one by one till the all the points come within the acceptable criteria.
Creatinine Linearity Difference
Creatinine Linearity Difference
Creatinine Linearity Difference
Creatinine Linearity Difference
Figures and tables from the paper
| NaOH Concentration(mmol/L) | Limit of Linearity (LOL) (mg/dl) | Slop (a) | Y-Intercept |
|---|---|---|---|
| 150 | 31.16 | 29.23 | -0.4578 |
| 250 | 23.77 | 28.27 | -0.6996 |
Table text extracted from the original PDF; superscript letters are shown inline. The original table image is below.
View original table image
References
2 in the original paper- NCCLS. Evaluation of the Linearity of Quantitative measurement Procedures: A Statastical Approach; Approved guideline. NCCLS document EP-6A [ISBN 1-56238-498-8]. NCCLS, 940 West Valley Road, Suite 1400, Wayne Pennsylvania 19087-1898USA, 2003.
- Edi. New Delhi: Elsevier, a division of reed Elsevier India Private Limited, 2012.