Rapid and complete paraffin removal from human tissue sections delivers enhanced Raman spectroscopic and histopathological analysis.

Incomplete removal of paraffin and organic contaminants from tissues processed for diagnostic histology has been a profound barrier to the introduction of Raman spectroscopic techniques into clinical practice. We report a route to rapid and complete paraffin removal from a range of formalin-fixed paraffin embedded tissues using super mirror stainless steel slides. The method is equally effective on a range of human and animal tissues, performs equally well with archived and new samples and is compatible with standard pathology lab procedures. We describe a general enhancement of the Raman scatter and enhanced staining with antibodies used in immunohistochemistry for clinical diagnosis. We conclude that these novel slide substrates have the power to improve diagnosis through anatomical pathology by facilitating the simultaneous combination of improved, more sensitive immunohistochemical staining and simplified, more reliable Raman spectroscopic imaging, analysis and signal processing.

Current Status of Microporous Metal-Organic Frameworks for Hydrocarbon Separations.

Separation of hydrocarbon mixtures into single components is a very important industrial process because all represent very important energy resources/raw chemicals in the petrochemical industry. The well-established industrial separation technology highly relies on the energy-intensive cryogenic distillation processes. The discovery of new materials capable of separating hydrocarbon mixtures by adsorbent-based separation technologies has the potential to provide more energy-efficient industrial processes with remarkable energy savings. Porous metal-organic frameworks (MOFs), also known as porous coordination polymers, represent a new class of porous materials that offer tremendous promise for hydrocarbon separations because of their easy tunability, designability, and functionality. A number of MOFs have been designed and synthesized to show excellent separation performance on various hydrocarbon separations. Here, we summarize and highlight some recent significant advances in the development of microporous MOFs for hydrocarbon separation applications.

Preliminary study on analysis and removal of wax from a Carrara marble statue.

This preliminary study has mainly focused on the wax identification by nuclear magnetic resonance (NMR) and removal. Wax is used for many purposes in the field of art as protective coatings on wooden, stone or metal objects. From the comparison of the spectra H NMR and in particular with the correspondence of the resonance peaks of the samples taken from the statue and beeswax and paraffin, we can conclude that the wax applied on the statue surface is beeswax. From our data, it can be concluded that, to remove the beeswax, from any stone support, the more effective solvent is the mixture of cyclohexane/ethyl acetate. The removal percentages ranged from 19 to 99%. Lower percentages of removal have been observed in the case of yellow marble, probably because of its high porosity. We can affirm that, this solvent mixture can be employed in real art objects using cotton swabs to remove protective wax.

Vortex-homogenized matrix solid-phase dispersion for the extraction of short chain chlorinated paraffins from indoor dust samples.

A simple and effective method for determining short chain chlorinated paraffins (SCCPs) in indoor dust is presented. The method employed a modified vortex-homogenized matrix solid-phase dispersion (VH-MSPD) prior to its detection by gas chromatography - electron-capture negative-ion mass spectrometry (GC-ECNI-MS) operating in the selected-ion-monitoring (SIM) mode. Under the best extraction conditions, 0.1-g of dust sample was dispersed with 0.1-g of silica gel by using vortex (2min) instead of using a mortar and pestle (3min). After that step, the blend was transferred to a glass column containing 3-g acidic silica gel, 2-g basic silica gel, and 2-g of deactivated silica gel, used as clean-up co-sorbents. Then, target analytes were eluted with 5mL of n-hexane/dichloromethane (2:1, v/v) mixture. The extract was evaporated to dryness under a gentle stream of nitrogen. The residue was then re-dissolved in n-hexane (10µL), and subjected to GC-ECNI-MS analysis. The limits of quantitation (LOQs) ranged from 0.06 to 0.25µg/g for each SCCP congener. Precision was less than 7% for both intra- and inter-day analysis. Trueness was above 89%, which was calculated by mean extraction recovery. The VH-MSPD combined with GC-ECNI-MS was successfully applied to quantitatively detect SCCPs from various indoor dust samples, and the concentrations ranged from 1.2 to 31.2µg/g.

Immunohistochemistry for Triple-Negative Breast Cancer.

Triple-negative breast cancers are a heterogeneous group of tumors that are, as yet, not entirely understood. Although triple-negative carcinomas are strictly defined as invasive carcinomas lacking expression of estrogen receptor, progesterone receptor, and HER2, some use the terms triple-negative and basal-like cancer synonymously. It should be noted that these are not entirely equivalent. Nevertheless, it has been shown that a panel of immunohistochemical markers can be used as a surrogate for genomic profiling and thus to identify basal-like breast cancers. We describe the panels of immunohistochemical markers that can be applied and how to interpret these markers herein.

Chemical composition of volatile and fixed oils from of Salvia argentea L. (Lamiaceae) growing wild in Sicily.

The chemical compositions of the essential oil and of the non-polar extracts (petroleum ether, dichloromethane) of the aerial parts (flowers, leaves and stems) of Salvia argentea L. were determined by GC-FID and gas chromatography-mass spectrometry analysis. 14-Hydroxy-α-humulene (40.1%) was recognised as the main constituents of the essential oil of S. argentea, together with 1,3,8-p-menthatriene (12.1%), globulol (7.4%) and ß-sesquiphellandrene (5.8%). Tritriacontane (9.9% and 14.1%), heptacosane (8.4% and 10.5%), hentriacontane (8.3% and 10.9%), tetradecanal (8.4% and 10.2%) and methyldotriacontane (7.9% and 7.6%) were recognised as the main constituents of the extracts in petroleum ether and dichloromethane, respectively, whereas methyl linolenate (36.6% and 13.5%) and methyl myristoleate (10.5% and 18.5%) were recognised as the main constituents of the methylated extracts.

Evaluation of different tissue de-paraffinization procedures for infrared spectral imaging.

In infrared spectral histopathology, paraffin embedded tissues are often de-paraffinized using chemical agents such as xylene and hexane. These chemicals are known to be toxic and the routine de-waxing procedure is time consuming. A comparative study was carried out to identify alternate de-paraffinization methods by using paraffin oil and electronic de-paraffinization (using a mathematical computer algorithm) and their effectiveness was compared to xylene and hexane. Sixteen adjacent tissue sections obtained from a single block of a normal colon tissue were de-paraffinized using xylene, hexane and paraffin oil (+ hexane wash) at five different time points each for comparison. One section was reserved unprocessed for electronic de-paraffinization based on a modified extended multiplicative signal correction (EMSC). IR imaging was carried out on these tissue sections. Coefficients based on the fit of a pure paraffin model to the IR images were then calculated to estimate the amount of paraffin remaining after processing. Results indicate that on average xylene removes more paraffin in comparison to hexane and paraffin oil although the differences were small. This makes paraffin oil, followed by a hexane wash, an interesting and less toxic alternative method of de-paraffinization. However, none of the chemical methods removed paraffin completely from the tissues at any given time point. Moreover, paraffin was removed more easily from the glandular regions than the connective tissue regions indicating a form of differential paraffin retention based on the histology. In such cases, the use of electronic de-paraffinization to neutralize such variances across different tissue regions might be considered. Moreover it is faster, reduces scatter artefacts by index matching and enables samples to be easily stored for further analysis if required.

Immobilization of Ag(i) into a metal-organic framework with -SO3H sites for highly selective olefin-paraffin separation at room temperature.

Introduction of Ag(i) ions into a sulfonic acid functionalized MOF ((Cr)-MIL-101-SO3H) significantly enhances its interactions with olefin double bonds, leading to its much higher selectivities for the separation of C2H4-C2H6 and C3H6-C3H8 at room temperature over the original (Cr)-MIL-101-SO3H and other adsorbents at room temperature.

Sample pretreatment optimization for the analysis of short chain chlorinated paraffins in soil with gas chromatography-electron capture negative ion-mass spectrometry.

Accurately quantifying short chain chlorinated paraffins (SCCPs) in soil samples with gas chromatograph coupled with electron capture negative ionization mass spectrometry (GC-ECNI-MS) is difficult because many other polychlorinated pollutants are present in the sample matrices. These pollutants (e.g., polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and toxaphene) can cause serious interferences during SCCPs analysis with GC-MS. Four main columns packed with different adsorbents, including silica gel, Florisil and alumina, were investigated in this study to determine their performance for separating interfering pollutants from SCCPs. These experimental results suggest that the optimum cleanup procedure uses a silica gel column and a multilayer silica gel-Florisil composite column. This procedure completely separated 22 PCB congeners, 23 OCPs and three toxaphene congeners from SCCPs. However, p,p'-DDD, cis-nonachlor and o,p'-DDD were not completely removed and only 53% of the total toxaphene was removed. This optimized method was successfully and effectively applied for removing interfering pollutants from real soil samples. SCCPs in 17 soil samples from different land use areas within a suburban region were analyzed with the established method. The concentrations of SCCPs in these samples were between 7 and 541 ng g(-1) (mean: 84 ng g(-1)). Similar homologue SCCPs patterns were observed between the soil samples collected from different land use areas. In addition, lower chlorinated (Cl(6/7)) C(10)- and C(11)- SCCPs were the dominant congeners.

Behavior, fate, and mass loading of short chain chlorinated paraffins in an advanced municipal sewage treatment plant.

Sewage treatment plants (STP) are an important source of short chain chlorinated paraffins (SCCPs) to the ambient environment through discharge of effluent and application of sludge. In this work, a field study was conducted to determine the behavior and possible removal of SCCPs during the sewage treatment process in an advanced municipal STP in Beijing, China. SCCPs were detected in all sewage water and sludge samples, and 97% of the initial mass loading in raw sewage was found to be associated with suspended matter. The total concentrations in raw influent, tertiary effluent, and dewatered sludge were 184 ± 19 ng/L, 27 ± 6 ng/L, and 15.6 ± 1.4 µg/g dry weight (d.w.), respectively. The dissolved concentrations of total SCCPs (∑SCCPs) significantly decreased during mechanical, biological, and chemical treatments. SCCP homologue profiles in aqueous phase were distinctly different from those in solid phase. Along the treatment process, the relative abundance of shorter chain and lower chlorinated congeners gradually increased in sewage water, but no obvious variations of homologue profiles were found in sludge. Mass flow analysis indicated, the removal efficiency in aqueous phase for ∑SCCPs was 82.2%, and the congener-specific removal efficiencies were positively related to their solid-water partition coefficients (K(d)). Mass balance results indicated that 0.8% and 72.6% of the initial SCCP mass loading were ultimately found in the effluents and dewatered sludge, respectively, while the remaining 26.6% was lost mainly due to biodegradation/biotransformation. It was suggested that the activated sludge system including basic anaerobic-anoxic-aerobic processes played an effective role in removing SCCPs from the wastewater, while the sorption to sludge by hydrophobic interactions was an important fate of SCCPs during the sewage treatment.

[Study on the chemical constituents of Pithecellobium clypearia].

OBJECTIVE: To study the chemical constituents of Pithecellobium clypearia. METHODS: Silica gel column chromatography and Sephadex LH-20 were used to separate and purify compounds from the EtOAc soluble fraction of Pithecellobium clypearia. 9 compounds were elucidated on the basis of physicochemical properties and spectrascopic analysis. RESULTS: They were identified as beta-Sitosterol (1), tritriacontane (2), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (3), oleanolic acid (4), 5,4'-dihydroxy-3,7,3'-trimethoxyflavone (5), alpha-amyrin (6), luteolin (7), ursolic acid (8), luteoloside (9). CONCLUSION: Compounds 3 and 5 are isolated from this plant for the first time.

Peptide extraction from formalin-fixed paraffin-embedded tissue.

This unit describes how to extract tryptic peptides from formalin-fixed, paraffin-embedded (FFPE) tissues for analysis using nano-reverse-phase liquid chromatography/tandem mass spectrometry (nRPLC-MS/MS). The tissues are deparaffinized in the first protocol. Following deparaffinization, the cells are harvested via one of two methods: needle dissection or laser capture microdissection (LCM). Needle dissection is performed using hydrated, unstained tissue, whereas LCM is performed with dehydrated, hematoxylin-stained tissue. Heat is applied to the collected cells to reverse the cross-links that have formed during the formalin fixation process. Finally, the cells are digested using filter-aided sample preparation, in which buffers are exchanged throughout the process. An alternate protocol using commercially available Liquid Tissue is also described. These samples are then ready for mass spectrometric analysis.

[Studies on the chemical constituents of Kalimeris indica].

OBJECTIVE: To study the chemical constituents from Kalimeris indica. METHODS: Compounds were isolated and purified with silica gel and Sephadex LH-20 column chromatography, their structures were determined by using spectroscopic analysis including MS and NMR. RESULTS: Nine compounds were isolated and identified as tritriacontane (I), hexadecanol (II), chrysophanol (III), vanillin (IV), physcion (V), beta-sitosterol (VI), stigmasterol (VII), leinoleic acid (VII), emodin (IX). CONCLUSION: All of them, except compound IV, VI, are isolated from this genus for the first time. Compound III, IV had the protective effects with the toxicity of CCl4 on primary cultered hepatocytes.

[Studies on the chemical constituents of Lonicera macranthoides].

OBJECTIVE: To study the chemical constituents of flower buds of Lonicera macranthoides. METHODS: The 90% EtOH extract of Lonicera macranthoides. was successively partitioned with petroleum ether and ethyl acetete. RESULTS: Repeated column chromatography of the ethyl acetete fraction afforded the following compounds (1-9): ginnol (1), triacontanol (2), ursolic acid (3), beta-sitosterol (4), triacontane (5), palmitic acid (6), beta-daucosterol (7), 3-decyl-3-octyldocosan-1-ol (8), 3-dodecyl-3-nonyldocosan-1-ol (9). All compounds except 4 are isolated from this plant for the first time while compounds 2, 3, 5, 8 and 9 are their first time been isolated from genus Lonicera.

[Studies on the chemical constituents from Inula cappa (II)].

OBJECTIVE: To study the chemical constituents of the root of Inula cappa. METHOD: The compounds were isolated and purified by recrystallization and chromatography with silica gel column and Sephadex LH-20 column. Their structures were identified by physico-chemical properties and spectral analysis. RESULTS: Seven compounds were isolated as scopolin (I), octacosanoic acid (II), tritriacontane (III), (2S,3S,4R,8E)-2-[(2'R)-2'-hydroxydocosanosylamino]-octadecane-1,3,4-triol(IV),(2S,3S,4R,8E)-2-[(2'R)-2'-hydroxytricosanosylamino]-octadecane-1,3,4-triol(V), (2S,3S,4R,8E)-2-[(2'R)-2'-hydroxytetracosanosylamino]-octadecane-1,3,4-triol(VI), (2S,3S,4R,8E)-2-[(2'R)-2'-hydroxypentacosanosylamino]-octadecane-1,3,4-triol(VIII), (2S,3S,4R,8E)-2-[(2'R)-2'-hydroxyhexacosanosylamino]-octadecane-1,3,4-triol(VIII). CONCLUSION: All compounds were isolated from Inula cappa for the first time.

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent.

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.

Separation of normal paraffins and evaluation of petroleum contamination in foods.

A method for separating n-paraffins from petroleum hydrocarbons in foods was developed. The method consists of 5 initial steps: digestion of sample with alkali, silica gel column chromatography, molecular sieve adsorption, destruction of the sieve with HCl, and oxidation with KMnO4. Recoveries of n-paraffins added to 55 g oyster at a level of 0.36 ppm ranged from 80% for normal pentadecane to 100% for n-paraffins over 18 carbon atoms. This method also facilitated the analysis of iso-paraffins such as pristane (2,6,10,14-tetramethylpentadecane) and phytane (2,6,10,14-tetramethylhexadecane), and other hydrocarbons, which are thought to be good marker compounds for the estimation of petroleum pollution.

Proteins and acids from petroleum.

The wax distillate fraction (boiling range 300 up to 400 degrees C) from the crude oil "El-Alameen" was found to be a good substrate for the biosynthesis of proteins and/or amino acids by bacteria under special culture conditions. The fermentation processes were accompanied by a refining effect to the oil fraction, elevating its refraction index and lowering its melting point, giving dewaxing effect to the oil fraction.