BACKGROUND: Simple manual method using a Nageotte hemocytometer counting residual leukocytes [White blood cells(WBC)] in blood components is subjective, labor-intensive, time consuming and variable within and between laboratories. The aim of this study was to evaluate usefulness for flow cytometric method to determinate very low numbers of leukocytes in leukocyte-free blood components. METHODS: Epics XL-MCL (Beckman Coulter Co.) was used for determination of fluorescence-labelled cells. The DNA in leukocytes was stained using propidium iodide (PI) and leukocytes were automatically analysed by flow cytometer. RESULTS: Linearity determined over a range of 0.5-50 WBC/muL was a value of r=0.994. The detection limit of this method was 4.4 WBC/muL and accuracy was 86.6% with linearity of r=0.991 over the 5-50 WBC/muL. Reproducibility was CV of 9.1% for 25.8 WBC/muL and 14.7% for 7.1 WBC/muL, respectively. CONCLUSION: Flow cytometric techniques provide a reproducible and objective tool for counting residual WBC in leukocyte free blood components compared with the Nageotte hemocytometer.
DNA ; Leukocytes* ; Limit of Detection ; Propidium

AIMTo establish a fluorospectrophotometric assay for the measurement of nitrite in blood.

METHODSInterference from hemoglobin and other blood ingredients was removed through sulfuric acid and phosphotungstic acid pretreatment. Fluorescence of 1-[H]-naphthotriazole from the reaction of 2,3-diaminonaphthalene with nitrite was determined with fluorospectrophotometry.

RESULTSThe following conditions were proper: Serum or plasma was treated with sulfuric acid and phosphotungstic acid pretreatment for two times, 2,3-diaminonaphthalene of 0.63 mmol x (L(-1)) was used, reaction solution pH and final pH were about 1.60 and 1.70 respectively, solution containing 2,3-diaminonaphthalene and supernatant after pretreatment was water-bathed at 20 degrees C for 15 minutes. The lower limit of detection was 24.27 nmol x L(-1). Nitrite determined in peripheral blood of healthy people was (10.91 +/- 2.38) micromol x L(-1), and its 95% distribution range was (6.24-15.57) micromol x L(-1).

CONCLUSIONIt's a relatively sensitive, specific, simple method. It's of some value to the study of nitric oxide.

A method of detecting lead was developed using square wave anodic stripping voltammetry (SWASV) with DNA-carbon nanotube paste electrode (CNTPE). The results indicated a sensitive oxidation peak current of lead on the DNA-CNTPE. The curves were obtained within a concentration range of 50 ngL-1-20 mgL-1 with preconcentration time of 100, 200, and 400 sec at the concentration of mgL-1, microgL-1, and ngL-1, respectively. The observed relative standard deviation was 0.101% (n = 12) in the lead concentration of 30.0 microgL-1 under optimum conditions. The low detection limit (S/N) was pegged at 8 ngL-1 (2.6 x10-8 M). Results showed that the developed method can be used in real-time assay in vivo without requiring any pretreatment and pharmaceutical samples, and food samples, as well as other materials requiring water source contamination analyses.
Electrodes ; Limit of Detection ; Nanotubes ; Plants* ; Water

OBJECTIVES: Rapid increase in engineered nanoparticles (ENPs) in many goods has raised significant concern about their environmental safety. Proper methodologies are therefore needed to conduct toxicity and exposure assessment of nanoparticles in the environment. This study reviews several analytical techniques for nanoparticles and summarizes their principles, advantages and disadvantages, reviews the state of the art, and offers the perspectives of nanometrology in relation to ENP studies. METHODS: Nanometrology is divided into five techniques with regard to the instrumental principle: microscopy, light scattering, spectroscopy, separation, and single particle inductively coupled plasma-mass spectrometry. RESULTS: Each analytical method has its own drawbacks, such as detection limit, ability to quantify or qualify ENPs, and matrix effects. More than two different analytical methods should be used to better characterize ENPs. CONCLUSIONS: In characterizing ENPs, the researchers should understand the nanometrology and its demerits, as well as its merits, to properly interpret their experimental results. Challenges lie in the nanometrology and pretreatment of ENPs from various matrices; in the extraction without dissolution or aggregation, and concentration of ENPs to satisfy the instrumental detection limit.
Limit of Detection ; Microscopy ; Nanoparticles ; Spectrum Analysis

BACKGROUND: Serum high density lipoprotein (HDL)-cholesterol level is used as an assessment of the risk of coronary heart disease. In this study, we evaluated direct measurement of HDL- cholesterol in serum with polyethylene-modified enzymes and sulfated alpha-cyclodextrin. METHODS: We evaluated the precision, the lower limit of detection, the recovery rate, the linearity, the interference for hemoglobin and the comparision with the result of HDL-cholesterol measured by selective precipitation method. We also studied the specificity of this direct method for very low density lipoprotein (VLDL) and low density lipoprotein (LDL). RESULTS: The total imprecision was 3.8% (low), 3.5% (middle), 3.2% (high). The lower limit of detection was 0 mg/L. The recovery rate was satisfactory. The linearity was also (r2=0.99). This method showed a good correlation (r2=0.97) with the selective precipitation method in HDL- cholesterol measurement. VLDL-cholesterol (up to 300 mg/L) increased HDL-cholesterol only less than 3% but increased VLDL-cholesterol to 400 mg/L, more than 750 mg/L caused 5% and 15% of overestimation of HDL-cholesterol, respectively. LDL-cholesterol (142-1,073 mg/L) increased or decreased HDL-cholesterol by some degree (about 15%). Hemoglobin (up to 3,000 mg/L) did not influence this assay. CONCLUSIONS: The direct measurement of HDL-cholesterol is satisfactory method in HDL- cholesterol measurement in good analytical performance and may be anticipated to reduce workload of laboratory because the sample pretreatment is not necessary.
Cholesterol* ; Coronary Disease ; Limit of Detection ; Lipoproteins* ; Sensitivity and Specificity

OBJECTIVEWe established a method of UPLC-MS/MS that was to detect fifteen precursors of perfluoroalkyl sulfonates (PFSA) and perfluoroalkyl carboxylates (PFCA) in serum.

METHODSBriefly, TBAS solution was added to sera, then the mixed solution was extracted with aliquots of MTBE. The MTBE aliquots were combined, evaporated to dryness under nitrogen, and reconsituted in 0.25 ml of methanol and water (1:1). Then the reconstituted solution through 0.2 µm nylon syringe filter was collected. Chromatographic separation was performed using a Waters ACQUITY (TM) BEH ¹⁸C column (50 mm × 2.1 mm × 1.7 mm). Analyte quantitation was performed in the negative electrospray ionization mode and multiple reaction monitoring (MRM).

RESULTSThree target substances, 6: 6PFPi, 6: 8PFPi, 8: 8PFPi, were externally confirmed by standard addition. Rates of recovery for these three chemicals were from 41.01% to 112.13% in two standard levels. And the relative standard deviations (RSD) were lower than 11.63% and higher than 1.80%. The other twelve substances were quantified with internal standard. Moreover in two standard levels, rate of recovery for these chemicals ranged from 70.25% to 127.51%. And RSD were more than 1.23% and less than 15.45%. And the corresponding limit of detection (LOD) and limit of quantitation (LOQ) for all target substances were 0.1-5.0 pg/ml and 0.2-10.0 pg/ml. Then we detected these target substances in ten different human serum samples. The levels of few substances were higher than LOD. And the ranges of FOSA-M, N-EtFOSA-M, N-MeFOSAA, N-EtFOSAA were respectively < LOD-0.94 pg/ml, < LOD-10.08 pg/ml, < LOD-6.74 pg/ml, < LOD-1.04 pg/ml.

CONCLUSIONThe method, with high sensitivity and accuracy, could meet the actual testing requirements.

BACKGROUND: C-reactive protein (CRP) concentrations are measured by two different assays depending on the clinical concern: the conventional CRP assay for estimating the extent of inflammation and the high sensitivity-CRP (hs-CRP) assay for assessing the risk of cardiovascular diseases. To integrate the conventional CRP test detecting acute phase response and the hs-CRP assay with a lower detection limit, we evaluated the performance characteristics of hs-CRP assay methods with a wide range of concentrations. METHODS: Immunonephelometric assay (BNII) and two turbidoimmunometric assays (TIA), the Hitachi 7600 with Daiichi reagent (Daiichi-Hitachi) and the Roche Cobas Integra 700 (Integra), were evaluated for the precision with 8 levels of pooled sera ranging from 0.3 to 120 mg/L and the agreement of TIA with the BNII assay using regression analysis and Bland-Altman analysis with 89 patient samples. RESULTS: The within-day coefficients of variation (CVs) of BNII were less than 10% in all levels of pooled sera. The CVs of Daiichi-Hitachi and Integra exceeded 10% in pooled sera below 1.0 mg/L and 0.5 mg/L, respectively. Both Daiichi-Hitachi and Integra appeared to be linear over the entire range of CRP concentrations used and were comparable with the results of BNII (Daiichi-Hitachi: y=0.98x+0.13, r=0.97, Integra; y=1.02x+0.22, r=0.99). However, at the concentrations over 100 mg/L TIA and BNII showed discrepant results. CONCLUSIONS: Both Daiichi-Hitachi and Integra showed a good precision over a wide range of CRP. However, the discrepant results found at very high concentrations require standardization among different assay methods or instruments.
C-Reactive Protein* ; Cardiovascular Diseases ; Humans ; Inflammation ; Limit of Detection

Salmonella are causative agents of gastroenteritis and systemic disease in animals. The invA gene was selected as a target sequence of loop-mediated isothermal amplification (LAMP) assay for diagnosis of Salmonella infection. The detection limits for broth dilution, spiked feces and enrichment were 10(4), 10(5) and 10(2) CFUs/mL, respectively. The LAMP assay developed in the present study may be a reliable method for detection of Salmonella spp. in pig feces.
Animals ; Diagnosis ; Feces* ; Gastroenteritis ; Limit of Detection ; Salmonella Infections ; Salmonella*

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) are used increasingly for tacrolimus monitoring. However, there are still variability of results due to home-brew reagents, so which cannot be warrantable data. We evaluated the analytical performance and clinical usefulness of a newly introduced MassTrak LC/MS/MS Tacrolimus kit (Waters Corporation, USA). METHODS: The performance of LC-MS/MS for determination of tacrolimus concentration were analyzed using patient samples and MassTrak LC/MS/MS Tacrolimus kit including calibrators, quality controls, internal standard, column and neat solution with respect to linearity, precision, lower limit of detection, lower limit of quantitation, sample carryover and comparison according to CLSI guidelines. The LC-MS/MS using home-brew reagents were performed for comparison test. RESULTS: The LC-MS/MS using MassTrak LC/MS/MS Tacrolimus kit showed a good linearity (R2> or =0.997) and precision (CV< 8%). Assigned LLOD (0.4 ng/mL) and LLOQ (0.8 ng/mL) were validated and carryover was estimated 0.5%. The system correlated well with the LC-MS/MS using home-brew reagents (R> or =0.974). CONCLUSIONS: LC-MS/MS using MassTrak LC/MS/MS Tacrolimus kit for determination of tacrolimus concentration showed good performance for linearity, precision, LLOD, LLOQ, carryover and comparison. Introduction of MassTrak LC/MS/MS Tacrolimus kit could be warranted results by manufacturer and useful for management of quality control.
Humans ; Indicators and Reagents ; Limit of Detection ; Mass Spectrometry ; Quality Control ; Tacrolimus

One-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using the MagNA Pure LC and LightCycler(TM) system was developed and validated for the detection and quantitation of hepatitis A virus (HAV) RNA. The assay was evaluated using in-house synthetic HAV RNA standard. The real-time RT-PCR assay could quantitate a dynamic range of HAV RNA standard between 10(2) and 10(8) copies per reaction. The regression coefficient of the standard curve was an 0.99. The detection limit of the assay was 31.3 RNA copies per reaction. The coefficient variations (CVs) of the assay in combination with automated RNA extraction were less than 1.91% in both intra- and inter-assay. The real-time RT-PCR assay for quantitative detection of HAV would serve a useful method for improving the safety of biological products.
Biological Products ; Hepatitis A virus* ; Hepatitis A* ; Hepatitis* ; Limit of Detection ; RNA*