Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

Simultaneous densitometric determination of eight food colors and four sweeteners in candies, jellies, beverages and pharmaceuticals by normal-phase high performance thin-layer chromatography using a single elution protocol.

The present work describes a novel application of high performance thin-layer chromatography for the separation and quantification of eight food colors (amaranth, allura red, erythrosine, fast green, indigo carmine, quinoline yellow, sunset yellow and tartrazine) and four intense sweeteners (acesulfame, aspartame, saccharin and neohesperidin) using a single mobile phase. The analyte zones were detected densitometrically at 210, 295, 450 and 550 nm. The best chromatographic performance for all the additives was achieved using acetonitrile: water: ethyl acetate: 10% aqueous ammonia (9:1:1:1, v/v/v/v) as the solvent system within 20 min. The developed method was validated as per International Conference on Harmonization (ICH) guidelines for linearity, sensitivity, selectivity, accuracy (recovery), precision (repeatability and reproducibility), robustness and stability. The limit of detection and limit of quantitation ranged from 0.2 to 9.8 ng/zone and 0.7-29.7 ng/zone respectively and the correlation coefficient ( r2) values for the calibration curves were ≥ 0.9973 for all the additives. The developed method was successfully applied to determine food colors and sweeteners in candies, jellies, beverages and pharmaceuticals with minimal sample processing. The recovery of different additives obtained from food products, beverages and pharmaceuticals ranged from 96.6 to 106.7 %. The results showed that all the samples contained food additives within the permissible limits. The developed method is simple, precise, accurate and economical for routine quality control of foodstuffs.

Measurement of Uncertainty for Aqueous Ethanol Wet-Bath Simulator Solutions Used with Evidential Breath Testing Instruments.

Aqueous ethanol wet-bath simulator solutions are used to perform calibration adjustments, calibration checks, proficiency testing, and inspection of breath alcohol instruments. The Toxicology Bureau of the New Mexico Department of Health has conducted a study to estimate a measurement of uncertainty for the preparation and testing of these wet-bath simulator solutions. The measurand is identified as the mass concentration of ethanol (g/100 mL) determined through dual capillary column headspace gas chromatography with flame ionization detector analysis. Three groups were used in the estimation of the aqueous ethanol wet-bath simulator solutions uncertainty: GC calibration adjustment, GC analytical, and certified reference material. The standard uncertainties for these uncertainty sources were combined using the method of root-sum-squares to give uc = 0.8598%. The combined standard uncertainty was expanded to U = 1.7% to reflect a confidence level of 95% using a coverage factor of 2. This estimation applies to all aqueous ethanol wet-bath simulator solution concentrations produced by this laboratory.