Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents.

The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.

Stability study of Azure B, Eosin Y and commercial Romanowsky Giemsa stock solutions using high performance liquid chromatography.

The stability of Azure B and Eosin Y in stock solutions of the individual compounds as well as in mixtures of the two dyes was studied. The purpose of the study of these two essential constituents of the Romanowsky Giemsa stain, commonly used in cytology and histology, was to select a stable mixture as a definitive stock solution. Two specific high performance liquid chromatographic methods were used to monitor qualitative and quantitative changes in solutions. Several parameters influencing the stability of Azure B were examined e.g. the type of counter ion, the presence of Eosin Y and the type of solvent used. The second part focused on the stability of Eosin Y in mixtures with different cationic dyes submitted to high temperatures. In conclusion, an Azure B SCN-Eosin Y acid mixture in dimethylsulphoxide (concentrations 0.75% and 0.12%, respectively) was selected as being the most appropriate composition of a stock solution for the Romanowsky Giemsa stain.

Quality control of albumin solutions by size-exclusion high-performance liquid chromatography, isoelectric focusing, and two-dimensional immunoelectrophoresis.

Quality control of albumin solutions for human use is of utmost importance because the presence of impurities can provoke adverse reactions in treated patients. Three different techniques were used to detect the presence of albumin oligomers and polymers as well as foreign proteins in commercial solutions. The relative concentrations of the former two were estimated using size-exclusion high-performance liquid chromatography. Isoelectric focusing and two-dimensional immunoelectrophoresis allowed detection of other contaminants of proteinaceous nature. The application of this combination of techniques supersedes the traditional approaches (gel filtration on polydextran gels, electrophoresis) in specificity and speed. Analysis of 34 lots of commercial albumin solutions from 22 manufacturers revealed the superior quality of preparations of placental rather than plasmatic origin.

Analysis of xanthene dyes by reversed-phase high-performance liquid chromatography on a polymeric column followed by characterization with a diode array detector.

A high-performance liquid chromatographic method on a polymeric column was developed for the analysis of xanthene dyes. The rigid polystyrene-divinylbenzene column was connected to a photodiode array detector to verify the identity and the purity of the dyes. For eosin Y a within-day precision of 1-2% was obtained, and on a day-to-day basis the coefficient of variation was 4.2%. The purity of commercial xanthene dyes was investigated, and the results show the divergence between the actual dye contents and the dye contents indicated on the label.

Comparison of cyclosporin A measurement in whole blood by six different methods.

In a multicentre trial, we analyzed and compared the cyclosporin concentrations in whole blood specimens (trough values) from renal and bone marrow transplant patients using six different methods: high-performance liquid chromatography (n-butyl reversed-phase column) as reference, and 5 immunoassay procedures using monoclonal specific or non-specific antibodies, or polyclonal antibodies, for measuring cyclosporin with and without its cross-reacting metabolites. Results obtained by the 2 specific RIAs show good correlations (r-values of 0.945 and 0.921) versus the HPLC method, with average assay ratios of: 1.52 for 3H-RIA/HPLC and 1.26 for 125I-RIA/HPLC. In contrast, ratios for non-specific immunoassay/HPLC show multiple-fold overestimations of cyclosporin with very wide variations: 4.58 for 3H-RIA/HPLC, 3.97 for 125I-RIA/HPLC and 3.87 for fluorescence polarization immunoassay (FPIA)/HPLC. These findings indicate 1) that the 'specific' 3H- and 125I-RIAs can be used for cyclosporin measurements in whole blood using normal therapeutic ranges established by HPLC, 2) that the important disparity and variable overestimations by 'non-specific' RIA or fluorescence polarization immunoassay, compared with HPLC, require adjustments in therapeutic ranges, and 3) that the available 'specific' and 'non-specific' immunoassays should enable establishment of within-house reference 'therapeutic/toxic' ranges for cyclosporin in individual centres, based on these 'specific' versus 'non-specific' assays.

Simultaneous determination of pentoxifylline and three metabolites in biological fluids by liquid chromatography.

We describe a sensitive and specific liquid-chromatographic assay for pentoxifylline and three of its metabolites in human plasma and urine. Addition of hydrochloric acid to the sample before extraction, and incorporation of acetic acid in the chromatographic eluent, allow the simultaneous determination of the four compounds plus an internal standard in one chromatographic run. Unlike gas-chromatographic procedures, this method does not involve derivatization no similar analysis of serum or plasma samples has been described before now. The method has been applied successfully to routine analysis and to pharmacokinetic studies.