Júlia Dénes

In vivo, in situ tissue analysis using rapid evaporative ionization mass spectrometry

The analysis of intact biological tissues by mass spectrometry (MS) has been pursued for more than three decades. However, mass spectrometric methods have always put strong constraints on the geometry and the preparation of these samples. Even with the recent advent of ambient ionization methods, not all of these restrictions have been lifted. MS analysis of biomolecules in tissue has traditionally been achieved by desorption ionization methods including secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption (MALDI), 19, 20] and desorption electrospray ionization (DESI) 5,18] methods. While desorption ionization methods are not appropriate for the analysis of vital (living) tissues, rapid thermal evaporation has the potential to establish the in situ, in vivo ionization of tissue constituents. The possible formation of organic ions from condensed-phase samples in a purely thermal process was initially proposed by Holland et al., and it was successfully demonstrated later. The rationale of rapid heating was to achieve molecular evaporation rates comparable to the rate of decomposition, which results in the formation of a considerable quantity of gaseous molecules or molecular ions. The quest for efficient thermal evaporation methods has led to the development of various thermally assisted ionization methods, including thermospray ionization. Since collisional cooling of nascent ions at higher pressure is more effective, thermal evaporation at atmospheric pressure is expected to suppress thermal decomposition. Atmospheric pressure thermal desorption ionization was demonstrated recently by the desorption of organic cations with minimal thermal degradation. 27] The present study is based on the discovery that rapid thermal evaporation of biological tissues yields gaseous molecular ions of the major tissue components, for example, phospholipids. As thermal evaporation of tissues is widely used in surgery (i.e., electrosurgery and laser surgery), it was sensible to use dedicated surgical instruments for the experiments. Combination of surgical and MS techniques also offers a possibility for in situ chemical analysis of tissue during surgery. Since the key feature of the technique is the fast evaporation of a sample, it was termed “Rapid Evaporative Ionization Mass Spectrometry” (REIMS). The tentative mechanism of ion formation is described in the Supporting Information. Electrosurgical dissection is based on the Joule heating and evaporation of tissues by an electric current. The presence of ionized water molecules during electrosurgical dissection raises the possibility of an alternative ionization mechanism involving neutral desorption and chemical ionization in the gas phase. For more details, see the Supporting Information. An electrosurgical electrode was used as an ion source coupled to a distant mass spectrometer employing a Venturi gas jet pump and 1–2 m long polytetrafluoroethylene (PTFE) tubing (Figure 1).

Identification of Biological Tissues by Rapid Evaporative Ionization Mass Spectrometry

The newly developed rapid evaporative ionization mass spectrometry (REIMS) provides the possibility of in vivo, in situ mass spectrometric tissue analysis. The experimental setup for REIMS is characterized in detail for the first time, and the description and testing of an equipment capable of in vivo analysis is presented. The spectra obtained by various standard surgical equipments were compared and found highly specific to the histological type of the tissues. The tissue analysis is based on their different phospholipid distribution; the identification algorithm uses a combination of principal component analysis (PCA) and linear discriminant analysis (LDA). The characterized method was proven to be sensitive for any perturbation such as age or diet in rats, but it was still perfectly suitable for tissue identification. Tissue identification accuracy higher than 97% was achieved with the PCA/LDA algorithm using a spectral database collected from various tissue species. In vivo, ex vivo, and post mortem REIMS studies were performed, and the method was found to be applicable for histological tissue analysis during surgical interventions, endoscopy, or after surgery in pathology.

Analysis of biological fluids by direct combination of solid phase extraction and desorption electrospray ionization mass spectrometry.

A novel, solid phase extraction (SPE)-based sample preparation method was developed for desorption electrospray ionization (DESI) mass spectrometry. Conventional SPE sample preparation was followed by a custom elution procedure. The eluate was evaporated from the closing frit of the cartridge using a gas jet. Thus the analyte was concentrated on the surface of the frit, which is ideal for DESI analysis. Application of the above SPE protocol allowed the concentration of the analyte content of up to 1 L liquid sample into a 1 mm diameter circular spot. The sample preparation procedure can improve the overall sensitivity of the method by up to 6 orders of magnitude if the sample volume is sufficient. The device has been tested using aqueous solutions of Rhodamine 116; the limit of detection was comparable to the LOD of electrospray analysis. Methodology was tested for drug monitoring applications in human serum. Levels of Cyclosporine A were determined using a 0.1 mL serum sample. Dynamic range of the method exceeded 3 orders of magnitude; the detection limit was below the therapeutic serum concentration of the drug.

In situ, real-time identification of biological tissues by ultraviolet and infrared laser desorption ionization mass spectrometry

Laser desorption ionization-mass spectrometric (LDI-MS) analysis of vital biological tissues and native, ex vivo tissue specimens is described. It was found that LDI-MS analysis yields tissue specific data using lasers both in the ultraviolet and far-infrared wavelength regimes, while visible and near IR lasers did not produce informative MS data. LDI mass spectra feature predominantly phospholipid-type molecular ions both in positive and negative ion modes, similar to other desorption ionization methods. Spectra were practically identical to rapid evaporative ionization MS (REIMS) spectra of corresponding tissues, indicating a similar ion formation mechanism. LDI-MS analysis of intact tissues was characterized in detail. The effect of laser fluence on the spectral characteristics (intensity and pattern) was investigated in the case of both continuous wave and pulsed lasers at various wavelengths. Since lasers are utilized in various fields of surgery, a surgical laser system was combined with a mass spectrometer in order to develop an intraoperative tissue identification device. A surgical CO(2) laser was found to yield sufficiently high ion current during normal use. The principal component analysis-based real-time data analysis method was developed for the quasi real-time identification of mass spectra. Performance of the system was demonstrated in the case of various malignant tumors of the gastrointestinal tract.

Real time analysis of brain tissue by direct combination of ultrasonic surgical aspiration and sonic spray mass spectrometry

Direct combination of cavitron ultrasonic surgical aspirator (CUSA) and sonic spray ionization mass spectrometry is presented. A commercially available ultrasonic surgical device was coupled to a Venturi easy ambient sonic-spray ionization (V-EASI) source by directly introducing liquified tissue debris into the Venturi air jet pump. The Venturi air jet pump was found to efficiently nebulize the suspended tissue material for gas phase ion production. The ionization mechanism involving solely pneumatic spraying was associated with that of sonic spray ionization. Positive and negative ionization spectra were obtained from brain and liver samples reflecting the primary application areas of the surgical device. Mass spectra were found to feature predominantly complex lipid-type constituents of tissues in both ion polarity modes. Multiply charged peptide anions were also detected. The influence of instrumental settings was characterized in detail. Venturi pump geometry and flow parameters were found to be critically important in ionization efficiency. Standard solutions of phospholipids and peptides were analyzed in order to test the dynamic range, sensitivity, and suppression effects. The spectra of the intact tissue specimens were found to be highly specific to the histological tissue type. The principal component analysis (PCA) and linear discriminant analysis (LDA) based data analysis method was developed for real-time tissue identification in a surgical environment. The method has been successfully tested on post-mortem and ex vivo human samples including astrocytomas, meningeomas, metastatic brain tumors, and healthy brain tissue.

Metabonomics of Newborn Screening Dried Blood Spot Samples: A Novel Approach in the Screening and Diagnostics of Inborn Errors of Metabolism

A novel, single stage high resolution mass spectrometry-based method is presented for the population level screening of inborn errors of metabolism. The approach proposed here extends traditional electrospray tandem mass spectrometry screening by introducing nanospray ionization and high resolution mass spectrometry, allowing the selective detection of more than 400 individual metabolic constituents of blood including acylcarnitines, amino acids, organic acids, fatty acids, carbohydrates, bile acids, and complex lipids. Dried blood spots were extracted using a methanolic solution of isotope labeled internal standards, and filtered extracts were electrosprayed using a fully automated chip-based nanospray ion source in both positive and negative ion mode. Ions were analyzed using an Orbitrap Fourier transformation mass spectrometer at nominal mass resolution of 100,000. Individual metabolic constituents were quantified using standard isotope dilution methods. Concentration threshold (cutoff) level-based analysis allows the identification of newborns with metabolic diseases, similarly to the traditional electrospray tandem mass spectrometry (ESI-MS/MS) method; however, the detection of additional known biomarkers (e.g., organic acids) results in improved sensitivity and selectivity. The broad range of detected analytes allowed the untargeted multivariate statistical analysis of spectra and identification of additional diseased states, therapeutic artifacts, and damaged samples, besides the metabolic disease panel.

Electrospray Post-Ionization Mass Spectrometry of Electrosurgical Aerosols

The feasibility of electrospray (ES) ionization of aerosols generated by electrosurgical disintegration methods was investigated. Although electrosurgery itself was demonstrated to produce gaseous ions, post-ionization methods were implemented to enhance the ion yield, especially in those cases when the ion current produced by the applied electrosurgical method is not sufficient for MS analysis. Post-ionization was implemented by mounting an ES emitter onto a Venturi pump, which is used for ion transfer. The effect of various parameters including geometry, high voltage setting, flow parameters, and solvent composition was investigated in detail. Experimental setups were optimized accordingly. ES post-ionization was found to yield spectra similar to those obtained by the REIMS technique, featuring predominantly lipid-type species. Signal enhancement was 20- to 50-fold compared with electrosurgical disintegration in positive mode, while no improvement was observed in negative mode. ES post-ionization was also demonstrated to allow the detection of non-lipid type species in the electrosurgical aerosol, including drug molecules. Since the tissue specificity of the MS data was preserved in the ES post-ionization setup, feasibility of tissue identification was demonstrated using different electrosurgical methods.

Identifying the margin: a new method to distinguish between cancerous and noncancerous tissue during surgery

part of The identification of tumor margins is crucially important in surgical oncology for ensuring a curative resection, accurate prognostication and for sparing of healthy tissues. This latter factor is particularly important in case of neurosurgery, where removal of cubic millimeters of brain tissue from eloquent areas could result in complete loss of psychomotor functions. Currently, tumor margins are established by means of preoperative medical imaging [1–3], and tumors are excised with a predefined safety zone or ‘resection margin’, which is defined by the anatomical location and the diagnosis of the primary tumor. Despite this approach, nearly 30% of breast cancers and 12% of colorectal cancers have an endangered resection margin. In other cases, where the tumor is approaching critically important anatomical features, such as major blood vessels or nerves, more marginal safety zones are used. (By contrast to general surgical approaches, neurosurgery does not apply a safety zone.) Perhaps more importantly, imaging techniques, such as CT scans or MRI, may understage tumors, leaving the surgeon to excise previously unrecognized advanced disease, and operative decisions are made without anatomical data on the disease state [4]. Staging laparoscopy has, therefore, become common place in gastric [5] and oesophogeal surgery, and cytology must be used to define peritoneal metastasis [6]. Furthermore, in the emergency setting, imaging techniques may poorly define the anatomy, and often preoperative biopsy data are not available to the surgeon. Finally, the principles of surgical oncology state that the tumor should not be incised or even handled so as to minimize the risk of metastasis; therefore, the surgeon may not even visualize the tumor itself during the course of the dissection. In the case of the excision of in situ cancers (e.g., anal intraepithelial neoplasia), the surgeon will be completely unaware of the tumor boundaries and will, in effect, perform a blind oncological excision.