"Repair cells" in equine uterine cytologic and histologic specimens

Cells resembling those known as "repair cells" in gynecologic cytology specimens from women were identified in uterine cytology specimens from infertile mares treated with antibiotics using indwelling uterine catheters. This prompted a study of the effect on the equine uterus of indwelling catheterization without antibiotic infusion, using light microscopic examination of cytologic and biopsy specimens and electron microscopic examination of biopsy specimens. Cytologic and biopsy specimens had features within normal limits at the start of the study. Following five days of indwelling catheterization, neutrophils were present in both cytologic and biopsy specimens. In cytologic specimens, numerous groups of "repair cells" were present; similar cells in biopsy specimens indicated this was a focal reaction. The large nuclei and prominent nucleoli of the "repair cells" suggested cellular proliferation or regeneration. However, this was contradicted by the ultrastructural sparsity of ribosomes, endoplasmic reticulum, Golgi apparatus and mitochondria. Inflammation and "repair cells" were not present in cytologic or biopsy specimens collected 40 days after the start of the study. Although these cells may be a component of a repair process, our results support the hypothesis that "repair cells" in human and equine gynecologic cytology specimens are injured, rather than regenerating, cells. The term dysphaneroplastic (Greek: "abnormal cytosol development") is proposed to describe these cells since the cytoplasm does not reflect the features of cellular activity suggested by the nuclear appearance.     

Simultaneous determination of the reducible and nonreducible cross-links of connective tissue. Analysis of mineralized and nonmineralized bone collagen

Secondary amine cross-links occur in collagen and elastin from a number of tissue sources. Quantification of these cross-links by amino acid analysis is complicated by the problem of separating cross-links, which are often minor components, from the more common amino acids and also because relatively large amounts of a cross-link are required to determine a color factor. A specific radioactive labeling method has been developed and used to quantify cross-links in bone collagen. Primary amines such as lysine and hydroxylysine are first guanidinated with 3,5-dimethylpyrazole-1-carboxamidine nitrate (DMPC). Secondary amines, which are unreactive with DMPC, are then quantitatively cyanoethylated with [14C]acrylonitrile. This procedure can be used to detect any secondary amine cross-link, with higher sensitivity than ninhydrin analysis, in peptide form as well as in acid hydrolysates. It is applied here in conjunction with [3H]NaBH4 reduction to simultaneously quantify Schiff base cross-links and amounts of in vivo reduction of Schiff bases in mineralized versus nonmineralized bovine bone.     

Photographic densitometry for quantitative DNA analysis of cytologic and histologic specimens.

We found that photographic densitometry (PD) is a useful technique for quantitative determinations of nuclear DNA content in clinical tumor material. Optimum conditions for the use of PD in clinical cytology and histopathology were worked out. A quantitative evaluation of the method was performed, particularly with respect to errors that may appear when measuring clinical tumor material. Our study showed that PD offers accurate DNA measurements in cytologic and histologic specimens. Ploidy level determinations in tumor cell populations in clinical material could be as accurately performed with PD as with scanning microspectrophotometry (SMP). Nuclear DNA content of individual cells as determined by PD correlated highly with nuclear DNA content determined by SMP (correlation coefficient, 0.96). Since the PD method is less influenced by background variation than are other image techniques (due to measurement of a photographic image), it is particularly useful in measurement of histopathologic sections, in which the background variation can introduce considerable errors. The method is also valuable with clinical cytologic smears, in which the presence of blood and other material disturbs the background. PD represents a valid complement to scanning microspectrophotometry and TV imaging systems, particularly for DNA analysis of tissue sections. Moreover, it can be applied easily in the clinical routine. Relevant tissue areas are selected and photographed by the pathologist or cytopathologist, and the measurement is performed by a laboratory technician.     

Kinetics of formation of metallic silver and binding of silver ions by tissue components.

The effect of time on the formation of metallic silver by tissue reducing groups follows a curve which can be divided into three main parts. In the first, which may last for several hours, the reaction is very slow, and only an undetectably small amount of metallic silver is produced. In the second period the speed of the reaction first increases in a progressive manner and then begins to decrease gradually; during the third period the speed approaches zero asymptotically. Binding of the silver ions by the tissue commences initially at its fastest rate; the level then decreases steadily to zero within about a quarter of an hour. There is no direct relationship between the amount of silver ion bound to the tissue and the formation of metallic silver. The latter cannot take place by way of direct (non-catalysed) reaction. The following mechanism is proposed for the process: Transfer of electrons from the reducing molecules to the silver ions is mediated at first by certain tissue sites (catalytic points) and then also by the steadily increasing total surface area of the metallic silver grains (autocatalysis). On the basis of this mechanism, several anomalies of both the argentaffin and argyrophil reactions are explained.     

Prognostic markers in histologic and cytologic specimens of breast cancer

OBJECTIVE: To correlate histologic and cytologic specimens of breast cancer by the expression of prognostic factors, such as estrogen receptor (ER), progesterone receptor (PR) and c-erbB-2, with immunochemical staining. STUDY DESIGN: Cytologic and histologic specimens from 83 patients were analyzed for expression of ER and PR, and 30 cases were analyzed for overexpression of c-erbB-2 using a standard immunochemical method. The material used for immunocytochemical staining was taken from the needle and syringe after each aspiration and smear preparation. The material was washed into a small container with preservative solution. Immunohistochemical staining was performed on paraffin-embedded specimens. RESULTS: A significant association was found between the histologic specimens and cytologic specimens by means of the expression of immunochemical markers. The best correlation between cytologic and histologic specimens was found when using c-erbB-2. CONCLUSION: A reliable and rapid evaluation of markers for breast cancer can be achieved by immunocytochemical staining on cytologic material. A good association was found between histologic and cytologic specimens using immunostaining.     

Cloud point extraction-flame atomic absorption spectrometry for pre-concentration and determination of trace amounts of silver ions in water samples

A cloud point extraction (CPE) method was used as a pre-concentration strategy prior to the determination of trace levels of silver in water by flame atomic absorption spectrometry (FAAS) The pre-concentration is based on the clouding phenomena of non-ionic surfactant, triton X-114, with Ag (I)/diethyldithiocarbamate (DDTC) complexes in which the latter is soluble in a micellar phase composed by the former. When the temperature increases above its cloud point, the Ag (I)/DDTC complexes are extracted into the surfactant-rich phase. The factors affecting the extraction efficiency including pH of the aqueous solution, concentration of the DDTC, amount of the surfactant, incubation temperature and time were investigated and optimized. Under the optimal experimental conditions, no interference was observed for the determination of 100 ng·mL⁻¹ Ag⁺ in the presence of various cations below their maximum concentrations allowed in this method, for instance, 50 μg·mL⁻¹ for both Zn²⁺ and Cu²⁺, 80 μg·mL⁻¹ for Pb²⁺, 1000 μg·mL⁻¹ for Mn²⁺, and 100 μg·mL⁻¹ for both Cd²⁺ and Ni²⁺. The calibration curve was linear in the range of 1–500 ng·mL⁻¹ with a limit of detection (LOD) at 0.3 ng·mL⁻¹. The developed method was successfully applied for the determination of trace levels of silver in water samples such as river water and tap water.     

Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography-mass spectrometry

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography-mass spectrometry (GC-MS) is described. Hair specimens were digested with 1M sodium hydroxide at 100 degrees C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with (S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC-MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1-45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios (R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of (R)-MA and (R)-AM than those of the corresponding (S)-enantiomers.     

Simultaneous determination of 3,4-dihydroxyphenylacetic acid and homovanillic acid in milligram amounts of rat striatal tissue by gas-liquid chromatography

Simultaneous determination of the major metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum has been achieved by gas-liquid chromatography. Striatal tissue from one rat was homogenized in IN HCl and one-tenth of the sample extracted with ethyl ether. After evaporation of the ether, the residue was reacted with a combination of 1-chloro-1,1,3,3,3-pentafluor-2-propanol and pentafluoropropionic anhydride followed by reaction with pentafluoropropionic anhydride. The derivatives were chromatographed on a 3% JXR column and quantitated using electron capture detection. The propionic homologs of DOPAC and HVA served as internal standards. The steady state levels of DOPAC and HVA were found to be 0.90 μg/gm±0.21 S.D. (N=12) and 0.66 μg/gm±0.16 S.D. (N=12) respectively.     

Factors affecting the formation of metallic silver and the binding of silver ions by tissue components

Summary The rate of formation of metallic silver has a maximum when plotted as a function of pH. The site of this maximum on a pH scale differs noticeably for various tissue elements. By contrast, the amount of silver ions bound to the tissue is a monotonously increasing function of the pH. A temperature rise decreases the length of the induction period and increases the gradient of the ascending section of the kinetic curve representing the formation of metallic silver. It also increases the maximum amount of silver ions bound to the tissue. An increase in the concentration (activity) of the silver ions in the impregnating bath has the same effect. Chemical composition and concentration of the complexing agent, as well as special ions in the impregnating bath to which earlier some definitive role has been attributed in the silver staining methods, proved to be ineffective when both pH and activity of silver ions were kept constant. Illumination of the reaction was also ineffective. The kinetic curves obtained in nonaqueous but polar media (e.g., acetone) exhibited the same qualitative characteristics as those obtained in aqueous solutions. No reaction between silver ions and tissue was observed in apolar solvents.     

Kinetics of formation of metallic silver and binding of silver ions by tissue components

Summary The effect of time on the formation of metallic silver by tissue reducing groups follows a curve which can be devided into three main parts. In the first, which may last for several hours, the reaction is very slow, and only an undetectably small amount of metallic silver is produced. In the second period the speed of the reaction first increases in a progressive manner and then begins to decrease gradually; during the third period the speed approaches zero asymptotically. Binding of the silver ions by the tissue commences initially at its fastest rate; the level then decreases steadily to zero within about a quarter of an hour. There is no direct relationship between the amount of silver ion bound to the tissue and the formation of metallic silver. The latter cannot take place by way of direct (non-catalysed) reaction. The following mechanism is proposed for the process: Transfer of electrons from the reducing molecules to the silver ions is mediated at first by certain tissue sites (catalytic points) and then also by the steadily increasing total surface area of the metallic silver grains (autocatalysis). On the basis of this mechanism, several anomalies of both the argentaffin and argyrophil reactions are explained.     

Optimal amounts of fluorescent dye improve expression microarray results in tumor specimens

Expression microarrays have great potential for clinical use but variability of the results represents a challenge for reliable practical application. The amount of fluorescent dye used in microarray experiments is a significant source of variability that has not been systematically studied. Here we demonstrate that the quantity of Cy3 dye affects microarray results performed on tumor specimens. Signal-to-noise ratios and coefficients of variation are significantly improved by increasing Cy3 to 150–180 pmol, but any further increase does not improve the data. In conclusion, optimal amounts of dye reduce variability and improve reliability of expression microarray experiments.     

A rapid liquid chromatographic determination of S-adenosylhomocysteine in subgram amounts of tissue.

A new assay is described for the activity of peptidyl-tRNA hydrolase on naturally occurring peptidyl-tRNA molecules. It takes advantage of the differential binding of the tRNA and peptide moieties to DEAE paper.     

Tissue identification and histologic study of six lung specimens from Egyptian mummies

Twenty-eight specimens obtained either from organ bundles in the body cavities of intact mummies, from damaged mummies, or from isolated canopic jars were examined for tissue identification and histopathologic study. The methods of rehydration and fixation were optimized by application to 40 dehydrated modern samples before studies of mummified tissue were undertaken. The tissue of origin could be definitely identified in 24 of the 28 specimens. Even small fragments obtained from isolated canopic jars proved suitable for histologic study. Six lung specimens were selected for more detailed study. All six showed focal deposition of anthracotic pigment. Electron diffraction and electron microprobe analysis of one of the small, polarizable crystals associated with the anthracosis indicated a mineral content of silica, aluminum, and iron. Two specimens showed focal areas of calcification consistent with old mycobacterial disease. Other histopathologic findings included evidence of pulmonary edema, emphysema, and pneumonia.