Julia Balog

Intraoperative Tissue Identification Using Rapid Evaporative Ionization Mass Spectrometry

A mass spectrometric approach was developed for intraoperative identification of cancerous tissue, in near–real-time. Diagnosing the Masses One of the best options for curing cancer is surgery. Yet, surgeons can leave cancerous tissue behind by not seeing the “tumor margins”—or edges of the tumor—clearly. If a surgeon isn’t sure whether tissue is normal or cancerous, the tissue is sent to a pathologist for testing. During this time (20 to 30 min), the patient remains under anesthesia, and, quite often, additional samples are required. To ensure that all malignant tissue is removed in the operating room, Balog and colleagues developed a mass spectrometry–based approach that identifies cancer during surgery. After analyzing ex vivo samples of cancerous, healthy, and benign/inflammatory tissue with rapid evaporative ionization mass spectrometry (REIMS), the authors created a database of the nearly 3000 tissue-specific mass spectra. These spectra were unique for each cancer type, with lipids such as phosphatidylcholine and phosphotidylinositol showing different ratios. Using these ratios, Balog et al. were even able to identify the origin of metastatic tumors ex vivo. To adapt this technology for use in vivo, during surgery, the authors created the “intelligent knife” (iKnife), which samples surgical smoke for mass spectrometric analysis. More than 800 spectra were acquired with the iKnife from 81 patients. These spectra, when matched against the previously created database, confirmed the results of normal histology, with low rates of false-positive and false-negative readouts. This first-in-human demonstration shows that the iKnife technology is ready for widespread use in the operating room to improve the accuracy of surgical intervention in cancer. Rapid evaporative ionization mass spectrometry (REIMS) is an emerging technique that allows near–real-time characterization of human tissue in vivo by analysis of the aerosol (“smoke”) released during electrosurgical dissection. The coupling of REIMS technology with electrosurgery for tissue diagnostics is known as the intelligent knife (iKnife). This study aimed to validate the technique by applying it to the analysis of fresh human tissue samples ex vivo and to demonstrate the translation to real-time use in vivo in a surgical environment. A variety of tissue samples from 302 patients were analyzed in the laboratory, resulting in 1624 cancerous and 1309 noncancerous database entries. The technology was then transferred to the operating theater, where the device was coupled to existing electrosurgical equipment to collect data during a total of 81 resections. Mass spectrometric data were analyzed using multivariate statistical methods, including principal components analysis (PCA) and linear discriminant analysis (LDA), and a spectral identification algorithm using a similar approach was implemented. The REIMS approach differentiated accurately between distinct histological and histopathological tissue types, with malignant tissues yielding chemical characteristics specific to their histopathological subtypes. Tissue identification via intraoperative REIMS matched the postoperative histological diagnosis in 100% (all 81) of the cases studied. The mass spectra reflected lipidomic profiles that varied between distinct histological tumor types and also between primary and metastatic tumors. Thus, in addition to real-time diagnostic information, the spectra provided additional information on divergent tumor biochemistry that may have mechanistic importance in cancer.

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.

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.

Characterization and Identification of Clinically Relevant Microorganisms Using Rapid Evaporative Ionization Mass Spectrometry

Rapid evaporative ionization mass spectrometry (REIMS) was investigated for its suitability as a general identification system for bacteria and fungi. Strains of 28 clinically relevant bacterial species were analyzed in negative ion mode, and corresponding data was subjected to unsupervised and supervised multivariate statistical analyses. The created supervised model yielded correct cross-validation results of 95.9%, 97.8%, and 100% on species, genus, and Gram-stain level, respectively. These results were not affected by the resolution of the mass spectral data. Blind identification tests were performed for strains cultured on different culture media and analyzed using different instrumental platforms which led to 97.8-100% correct identification. Seven different Escherichia coli strains were subjected to different culture conditions and were distinguishable with 88% accuracy. In addition, the technique proved suitable to distinguish five pathogenic Candida species with 98.8% accuracy without any further modification to the experimental workflow. These results prove that REIMS is sufficiently specific to serve as a culture condition-independent tool for the identification and characterization of microorganisms.

In Vivo Endoscopic Tissue Identification by Rapid Evaporative Ionization Mass Spectrometry (REIMS)

Gastrointestinal cancers are a leading cause of mortality, accounting for 23 % of cancer-related deaths worldwide. In order to improve outcomes from these cancers, novel tissue characterization methods are needed to facilitate accurate diagnosis. Rapid evaporative ionization mass spectrometry (REIMS) is a technique developed for the in vivo classification of human tissue through mass spectrometric analysis of aerosols released during electrosurgical dissection. This ionization technique was further developed by utilizing surface induced dissociation and was integrated with an endoscopic polypectomy snare to allow in vivo analysis of the gastrointestinal tract. We tested the classification performance of this novel endoscopic REIMS method in vivo. It was shown to be capable of differentiating between healthy layers of the intestinal wall, cancer, and adenomatous polyps based on the REIMS fingerprint of each tissue type in vivo.

Identification of the Species of Origin for Meat Products by Rapid Evaporative Ionization Mass Spectrometry

Increasingly abundant food fraud cases have brought food authenticity and safety into major focus. This study presents a fast and effective way to identify meat products using rapid evaporative ionization mass spectrometry (REIMS). The experimental setup was demonstrated to be able to record a mass spectrometric profile of meat specimens in a time frame of <5 s. A multivariate statistical algorithm was developed and successfully tested for the identification of animal tissue with different anatomical origin, breed, and species with 100% accuracy at species and 97% accuracy at breed level. Detection of the presence of meat originating from a different species (horse, cattle, and venison) has also been demonstrated with high accuracy using mixed patties with a 5% detection limit. REIMS technology was found to be a promising tool in food safety applications providing a reliable and simple method for the rapid characterization of food products.

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.

Rapid Evaporative Ionisation Mass Spectrometry (REIMS) Provides Accurate Direct from Culture Species Identification within the Genus Candida.

Members of the genus Candida, such as C. albicans and C. parapsilosis, are important human pathogens. Other members of this genus, previously believed to carry minimal disease risk, are increasingly recognised as important human pathogens, particularly because of variations in susceptibilities to widely used anti-fungal agents. Thus, rapid and accurate identification of clinical Candida isolates is fundamental in ensuring timely and effective treatments are delivered. Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has previously been shown to provide a high-throughput platform for the rapid and accurate identification of bacterial and fungal isolates. In comparison to commercially available matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF), REIMS based methods require no preparative steps nor time-consuming cell extractions. Here, we report on the ability of REIMS-based analysis to rapidly and accurately identify 153 clinical Candida isolates to species level. Both handheld bipolar REIMS and high-throughput REIMS platforms showed high levels of species classification accuracy, with 96% and 100% of isolates classified correctly to species level respectively. In addition, significantly different (FDR corrected P value < 0.05) lipids within the 600 to 1000 m/z mass range were identified, which could act as species-specific biomarkers in complex microbial communities.