scholarly journals Development of an Automated Enzymatic Method to Quantify Pyruvate Kinase in Red Blood Cells

2019 ◽  
Vol 5 (1) ◽  
pp. 54-61
Author(s):  
Jun Lu ◽  
David G Grenache
Keyword(s):  
Linear Regression ◽  
Red Blood Cells ◽  
Pyruvate Kinase ◽  
Blood Cells ◽  
Limit Of Detection ◽  
Linear Regression Analysis ◽  
Method Comparison ◽  
Reference Interval ◽  
Automated Method ◽  
K Factor

Abstract Background Pyruvate kinase (PK) deficiency is the most common cause of nonspherocytic hemolytic anemia owing to defective glycolysis. This study developed and validated an automated method to measure PK activity in red blood cells (RBCs). Methods PK catalyzes the reaction of phosphoenolpyruvate with ADP to form pyruvate and ATP. The pyruvate is reduced in the presence of lactate dehydrogenase and NADH to produce lactate and NAD+. The rate of absorbance decrease at 340 nm is proportional to PK activity. PK and hemoglobin (Hb) measurements were performed on a Roche cobas c501 analyzer. After establishing a k-factor, accuracy, linearity, imprecision, sensitivity, and stability were validated and the reference interval was verified. Results The k-factor was −9477. Accuracy was evaluated by method comparison (n = 56). Linear regression yielded y = 1.0x − 0.57, and R2 of 0.93. Linearity was determined by combining a high sample with hemolyzing solution in 6 different ratios. Linear regression analysis yielded y = 1.02x − 2.68, and R2 of 1.0. The assay was linear to 87 U/dL. Precision was evaluated by testing hemolysates in 3 replicates/day for 10 days. Within-run imprecision was 1.9% and 2.5% and total imprecision was 4.0% and 5.6% at 14.0 and 8.1 U/g Hb, respectively. The limit of blank was 0.0, and the limit of detection was 1.0 U/dL. Stability was determined in 4 sample types at 3 different temperatures; the changes were all <10% when compared with t0. The current PK reference interval of 4.6 to 11.2 U/g Hb was verified. Conclusions This automated assay for quantifying PK in RBCs has acceptable performance characteristics and is fit for intended use.

scholarly journals Direct and rapid measurement of hydrogen peroxide in human blood using a microfluidic device

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
R. Gaikwad ◽  
P. R. Thangaraj ◽  
A. K. Sen
Keyword(s):  
Hydrogen Peroxide ◽  
Microfluidic Device ◽  
Human Blood ◽  
Blood Cells ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Time Interval ◽  
Fluorescence Measurement ◽  
Rapid Measurement ◽  
Automated Method

AbstractThe levels of hydrogen peroxide ($${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 ) in human blood is of great relevance as it has emerged as an important signalling molecule in a variety of disease states. Fast and reliable measurement of $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 levels in the blood, however, continues to remain a challenge. Herein we report an automated method employing a microfluidic device for direct and rapid measurement of $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 in human blood based on laser-induced fluorescence measurement. Our study delineates the critical factors that affect measurement accuracy—we found blood cells and soluble proteins significantly alter the native $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 levels in the time interval between sample withdrawal and detection. We show that separation of blood cells and subsequent dilution of the plasma with a buffer at a ratio of 1:6 inhibits the above effect, leading to reliable measurements. We demonstrate rapid measurement of $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 in plasma in the concentration range of 0–49 µM, offering a limit of detection of 0.05 µM, a sensitivity of 0.60 µM−1, and detection time of 15 min; the device is amenable to the real-time measurement of $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 in the patient’s blood. Using the linear correlation obtained with known quantities of $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 , the endogenous $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 concentration in the blood of healthy individuals is found to be in the range of 0.8–6 µM. The availability of this device at the point of care will have relevance in understanding the role of $${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$ H 2 O 2 in health and disease.

scholarly journals Pre-analytical quality control in hemostasis laboratories: visual evaluation of hemolysis index alone may cause unnecessary sample rejection

2019 ◽  
Vol 43 (2) ◽  
pp. 67-76
Author(s):  
Simona Storti ◽  
Elena Battipaglia ◽  
Maria Serena Parri ◽  
Andrea Ripoli ◽  
Stefania Lombardi ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Linear Regression ◽  
Visual Inspection ◽  
Clinical Chemistry ◽  
Linear Regression Analysis ◽  
Reference Method ◽  
Analytical Performance ◽  
Visual Evaluation ◽  
Automated Method ◽  
Visual Index

Abstract Background Visual inspection is the most widespread method for evaluating sample hemolysis in hemostasis laboratories. The hemolysis index (HI) was determined visually (visual index, VI) and measured on an ACL TOP 750 (IL Werfen) system with a hemolysis-icterus-lipemia index (HIL) module. These values were compared with those measured on clinical chemistry systems Unicel DXC600 and AU680 and with quantitation of free-hemoglobin (Hb) performed by a spectrophotometric measurement method (SMM). Methods The HI was measured in 356 sodium citrate plasma samples, 306 of which were visibly hemolyzed to varying degrees and 50 were not hemolyzed. The analytical performance of each method was evaluated. Results Linear regression analysis, calculated between SMM and the other systems in the study, returned coefficients of determination r2 = 0.853 (AU680), r2 = 0.893 (DXC600) and r2 = 0.917 (ACL TOP 750). An r2 = 0.648 was obtained for linear regression analysis between VI and ACL TOP 750. In addition, ACL TOP 750 showed an excellent correlation in multivariate analysis (r2 = 0.958), showing good sensitivity (0.939) and specificity (0.934) and a diagnostic accuracy of 94%. By comparison, DXC600 and AU680 showed coefficients of determination of 0.945 and 0.923, respectively. A cut-off was set at 0.15 g/L free-Hb, as determined by the automated method, such that any hemostasis samples measuring above this threshold should not be analyzed. Based on this criterion, samples were classified as accepted or rejected, and the number of samples discarded during VI or ACL TOP 750 measurements was compared. Conclusions This study confirmed that hemostasis laboratories should consider introducing an objective, automated and standardized method to check samples for hemolysis. By relying solely on visual inspection, up to 50% of samples could be unnecessarily rejected. The ACL TOP 750 system demonstrated a satisfactory analytical performance, giving results comparable to those of the reference method.

scholarly journals Evaluation of Extra Virgin Olive Oil Adulteration with Edible Oils using ATR-FTIR Spectroscopy

2020 ◽  
Vol 5 (1) ◽  
pp. 35-44
Author(s):  
Nuraznee Mashodi ◽  
Nurul Yani Rahim ◽  
Norhayati Muhammad ◽  
Saliza Asman
Keyword(s):  
Linear Regression ◽  
Olive Oil ◽  
Ftir Spectroscopy ◽  
Corn Oil ◽  
Edible Oils ◽  
Limit Of Detection ◽  
Linear Regression Analysis ◽  
Virgin Olive Oil ◽  
Extra Virgin Olive Oil ◽  
Regression Graph

Extra virgin olive oil (EVOO) is categorized as expensive oil due to high-quality nutritional value. Unfortunately, EVOO is easily adulterated with other low-quality edible oils. Therefore, this study was done to differentiate and analyze the adulteration of EVOO with other edible oils using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The study was used several edible oils included canola oil, corn oil, sunflower oil, and soybean oil as an adulterant for EVOO. The adulterant EVOO samples were prepared by mixing with dissimilar concentrations of the solely edible oils (20 %, 40 %, 60 % and 80 % (v/v)). The main functional groups of EVOO and other edible oils are O-H, C-H, C=C and C=O groups were assigned around 3500 cm-1, 2925 cm-1, 3006 cm-1 and 1745 cm-1 wavenumbers, respectively. From the comparison of EVOO and other adulterant edibles oil spectra, it showed that the EVOO has the lowest absorbance intensity at around 3006 cm-1 represented double bond which is closely related to the composition of oil sample. The adulteration of EVOO was evaluated by analysing the changes in the absorbance based on the linear regression analysis graph of the bands at 3006 and 2925 cm-1 and the limit of detection (LOD) was measured. The graph of A3008/A2925 with good relative coefficients (R2) and lower LOD is more favourable than the linear regression graph of A3006 versus percentage of edible oils added in EVOO. This study showed that ATR-FTIR spectroscopy is a convenient tool for analysing the adulteration of EVOO.

scholarly journals Direct and Rapid Measurement of Hydrogen Peroxide in Human Blood Using a Microfluidic Device

2020 ◽  
Author(s):  
Ravindra Gaikwad ◽  
Paul Thangaraj ◽  
Ashis Sen
Keyword(s):  
Hydrogen Peroxide ◽  
Microfluidic Device ◽  
Human Blood ◽  
Blood Cells ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Time Interval ◽  
Fluorescence Measurement ◽  
Rapid Measurement ◽  
Automated Method

Abstract The levels of hydrogen peroxide (H2O2) in human blood is of great relevance as it has emerged as an important signalling molecule in a variety of disease states. Fast and reliable measurement of H2O2 levels in the blood, however, continues to remain a challenge. Herein we report an automated method employing a microfluidic device for direct and rapid measurement of H2O2 in human blood based on laser-induced fluorescence measurement. Our study delineates the critical factors that affect measurement accuracy – we found blood cells and soluble proteins significantly alter the native H2O2 levels in the time interval between sample withdrawal and detection. We show that separation of blood cells and subsequent dilution of the plasma with a buffer at a ratio of 1:6 inhibits the above effect, leading to reliable measurements. We demonstrate rapid measurement of in plasma in the concentration range of 0 – 49 µM, offering a limit of detection of 0.05 µM, a sensitivity of 0.60 µM-1, and detection time of 15 min; the device is amenable to the real-time measurement of H2O2 in the patient’s blood. Using the linear correlation obtained with known quantities of H2O2, the endogenous H2O2 concentration in the blood of healthy individuals is found to be in the range 2 – 6 µM. The availability of this device at the point of care will have relevance in understanding the role of H2O2 in health and disease.

scholarly journals Continuous changes in biological levels of complete blood count in a high altitude area of China

2020 ◽  
Vol 8 ◽  
pp. 205031212093133 ◽  
Author(s):  
Xuan Luo ◽  
Lei Feng ◽  
XueJing Bai ◽  
JiangXian Zhu ◽  
GuanCheng Zhao ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Complete Blood Count ◽  
White Blood Cells ◽  
Age Groups ◽  
Reference Interval ◽  
Distribution Width ◽  
Men And Women ◽  
Cell Parameters

Objectives: We aimed to establish a new reference interval of blood cell parameters by classifying and counting blood Cells of 16- to 85-year-old healthy volunteers and observing continuous changes with age. Methods: We analyzed the blood cell parameters of 42,678 cases (men, 24,406; women, 18,272), and compared the blood cell parameters of men and women in different age groups using an independent samples t-test. Using limits of 2.5%–97.5%, a 90% confidence interval was used to develop new reference intervals. Results: Counts of blood cell parameters, including white blood Cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood Cells, hemoglobin, hematocrit, distribution width of red blood Cells and platelets, were found to differ between men and women in different age groups. These parameters were used to establish a new reference interval of blood Cells. Conclusion: The blood cell parameters of both men and women changed with increasing age. The reference interval that we established will provide more accurate basic evidence for clinical diagnosis and treatment of diseases.

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