A self-established method for the quantitative determination of plasma free hemoglobin utilizing the hemolysis index of Siemens ADVIA 2400 chemistry system

Materials

The study was approved by the Institutional Ethics Committee of Hangzhou Traditional Chinese Medicine Hospital, Affiliated with Zhejiang Chinese Medical University. Blood samples used in the experiments were obtained from residual whole blood material collected using ethylenediaminetetraacetic acid-K2 anticoagulant tubes. All samples were stored at 4°C for a maximum duration of 7 days, if necessary.

Instrumentation

The measurement of plasma HI was conducted using the Siemens ADVIA 2400 chemistry system (Siemens Healthcare GmbH, Erlangen, Germany). The HI was set to quantitative mode, which measured the absorbance of plasma diluted by a factor of 4.2 (25 μL sample volume + 80 μL 0.9% saline solution) at wavelengths of 571 nm and 658 nm relative to a 0.9% saline solution and calculated using an embedded preset algorithm. The absorbance of hemolysis was adjusted in the presence of lipemia as follows: Corrected hemolysis absorbance = hemolysis absorbance - 1.1 × lipemia absorbance, while icterus theoretically did not exhibit interference at 571 nm.^[8]

Preparation of standard hemoglobin solutions and quality control (QC) samples

Erythrocytes were enriched through centrifugation at 1000 g from a whole blood sample. The enriched cells were then diluted with distilled water containing a final concentration of 0.1% Triton-X100 (Sigma-Aldrich Corporation, St. Louis, America). The solution was subsequently blended using an oscillating blender to ensure thorough rupture of erythrocytes, followed by centrifugation (12000 g, 10 min) for the removal of cellular debris. The supernatant was collected as a stock solution of hemoglobin, and its concentration was determined through five repeated assays using the Sysmex XN-1000 analyzer (Sysmex America, IL, USA).

Due to the lack of reference material, the hemoglobin standard solution, calibrator, and QC samples were diluted from the stock solution with artificial plasma. The hemolysis plasma samples were stored at −20℃ until analysis and underwent a single thawing process.^[9]

Upper limit of detection (ULoD)

The clinical chemistry system has a detection capability up to an optical density (OD) value of 2.5. The ULoD capability was assessed using a standard solution containing approximately 200 g/L of hemoglobin. This standard solution underwent a double ratio dilution method employing saline. Subsequently, the diluted solutions were evaluated using the HI assay, and the corresponding OD values were observed.

Calibration

To establish the standard curve for the relationship between hemoglobin concentration and quantitative HI results, a hemoglobin calibrator was prepared with a concentration set close to half of the ULoD. The ADVIA 2400 system was used to measure the HI of both the calibrator and saline (considered as 0 g/L hemoglobin) 3 times. The average of HI results was plotted on the X-axis against their corresponding hemoglobin concentrations on the Y-axis to construct the standard curve.

Linearity evaluation

To evaluate linearity in accordance with the Clinical and Laboratory Standards Institute (CLSI) protocol EP06,^[10] a standard hemoglobin solution near its upper detection limit was progressively diluted with saline to generate samples containing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and full concentration of the standard hemoglobin solution. The regression equation for hemoglobin concentration in relation to the HI value was derived through correlation analysis. The threshold for the coefficient of determination (R²) was set at a minimum value of >0.995.

Limit of blank (LoB) and lower limit of detection (LLoD)

The LoB and the LLoD were evaluated following the CLSI protocol EP17-A2.^[11] The LoB and LLoD were determined by analyzing five blank samples (saline solution) and five low-level samples (equivalent to 10 times the estimated LoB), which were run on 7 different days, with each sample replicated twice.

Lower limit of quantification (LLoQ)

The LLoQ was determined by conducting repeat analyses on five hemoglobin samples with a low concentration of 0.085 g/L over the course of 3 separate days. Each sample was tested 4 times per day. To ensure accuracy, a coefficient of variation (CV%) below 10% was employed, aligning with both our expectations and experiences shared by peers.^[7]

Precision evaluation

The precision (CV%) was evaluated following the 20 × 2 × 2 experimental designs in accordance with the CLSI-EP05-A3 guideline.^[12] Therefore, four levels of QCs were measured over a span of 20 testing days, with two runs conducted per day and two replicates performed per run. The measurement repeatability and within-laboratory precision were calculated.

Trueness evaluation (method comparison)

According to the CLSI protocol EP09C-A3,^[13] the trueness performance was evaluated by comparing samples of varying grades of hemolysis (n = 40) with the Sysmex XN-1000 analyzer, which could be traced back to the cyanmethemoglobin method. The results were compared using linear regression analysis based on the nonparametric Passing and Bablok method, and system differences were analyzed according to Bland and Altman. ^[14,15]

Statistical analysis

Microsoft Excel was used for analyzing variance, while MedCalc (Version 20.216) was employed for conducting least-squares linear regression analysis.

ULoD and calibration curve

The OD value of 2.08 was observed for a hemoglobin concentration of 24.75 g/L, suggesting an approximate upper limit for detectable hemoglobin concentration at around 29 g/L. Consequently, a hemoglobin calibrator was prepared at a level of 13.8 g/L. The calibration curve was determined by employing linear regression analysis, resulting in the derived equation y = 0.009x.

Detection capability and linearity

The non-parametric method was selected to estimate LoB and LLoD because the measured results exhibited an abnormal distribution in terms of variability.^[16] The LoB was determined to be 0.005 g/L, while the LLoD was found to be 0.050 g/L. The LLoQ was established at 0.085 g/L. The HI method showed excellent linearity (determination coefficient R² = 0.9998, P < 0.0001) across the range of 0–25.2 g/L, with a regression equation describing the relationship between the estimated and the measured fHb concentrations as y = 0.9579x + 0.0369. Thus, our self-established method demonstrates an analytical measurement range of 0.085~25.2 g/L for detecting fHb [Figure 1].

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Figure 1:

The linearity of the self-established method was investigated by diluting a standard hemoglobin solution (28 g/L) with saline. The measured concentrations of hemoglobin in the diluted samples were plotted against their expected values. (First order regression analysis, y = 0.9579x + 0.0369, R² = 0.9998).

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Precision and trueness

The self-established method exhibited a total CV of 0.47%, 0.84%, 0.95%, and 0.54% at concentrations of approximately 22 g/L, 11 g/L, 5.5 g/L, and 2.5 g/L of fHb, respectively [Table 1].

The Passing and Bablok regression analysis and Bland-Altman plot are depicted in Figure 2. The regression analysis showed an excellent correlation and satisfactory agreement between the two methods, as indicated by the slope (1.04; 95% confidence interval [CI], 1.03~1.05) and intercept (−0.10; 95% CI, −0.19~−0.03). Spearman’s rank correlation coefficient was found to be r = 1 (P < 0.0001). The bias plot revealed slightly lower values for the HI assay (0.35 g/L).

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Figure 2:

Method comparison was conducted between the self-established method and Sysmex XN-1000 for determining the concentrations of fHb in 40 samples. (a) the Bland-Altman bias plot, (Blue line: mean bias value; Red lines: 1.96 standard deviation) and (b) regression analysis by the Passing-Bablok method (Solid line: The regression line; Dashed lines: Confidence interval for the regression line; Dotted line: x=y).

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