Non-invasive screening of breast cancer from fingertip smears-a proof of concept study.

Breast cancer is a global health issue affecting 2.3 million women per year, causing death in over 600,000. Mammography (and biopsy) is the gold standard for screening and diagnosis. Whilst effective, this test exposes individuals to radiation, has limitations to its sensitivity and specificity and may cause moderate to severe discomfort. Some women may also find this test culturally unacceptable. This proof-of-concept study, combining bottom-up proteomics with Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) detection, explores the potential for a non-invasive technique for the early detection of breast cancer from fingertip smears. A cohort of 15 women with either benign breast disease (n = 5), early breast cancer (n = 5) or metastatic breast cancer (n = 5) were recruited from a single UK breast unit. Fingertips smears were taken from each patient and from each of the ten digits, either at the time of diagnosis or, for metastatic patients, during active treatment. A number of statistical analyses and machine learning approaches were investigated and applied to the resulting mass spectral dataset. The highest performing predictive method, a 3-class Multilayer Perceptron neural network, yielded an accuracy score of 97.8% when categorising unseen MALDI MS spectra as either the benign, early or metastatic cancer classes. These findings support the need for further research into the use of sweat deposits (in the form of fingertip smears or fingerprints) for non-invasive screening of breast cancer.

Exploring the problem of determining human age from fingermarks using MALDI MS-machine learning combined approaches.

For over a century fingerprints have been predominantly used as a means of biometric identification. Notwithstanding, the unique pattern of lines that can contribute to identifying a suspect is made up of molecules originating from touch chemistry (contaminants) as well as from within the body. It is the latter class of molecules that could provide additional information about a suspect, such as lifestyle, as well as physiological, pharmacological and pathological states. An example of the physiological state (and semi-biometric information) is the sex of an individual; recent investigations have demonstrated the opportunity to determine the sex of an individual with an 86% accuracy of prediction based on the peptidic/protein profile of their fingerprints. In the study presented here, the first of its kind, a range of supervised learning predictive methods have been evaluated to explore the depth of the issue connected to human age determination from fingermarks exploiting again the differential presence of peptides and small proteins. A number of observations could be made providing (i) an understanding of the more appropriate study design for this kind of investigation, (ii) the most promising prediction model to test within future work and (iii) the deeper issues relating to this type of determination and concerning a mismatch between chronological and biological ages. Particularly resolving point (iii) is crucial to the success in determining the age of an individual from the molecular composition of their fingermark.

Application of MALDI MS imaging after sequential processing of latent fingermarks.

Latent fingermarks are routinely visualised by subjecting them to one or more CSI/crime lab processes to maximise the recovery of ridge flow and minutiae permitting an identification. In the last decade mass spectrometric imaging (MSI) techniques have been applied to fingermarks to provide information about a suspect and/or on the circumstances of the crime as well as yielding additional images of the ridge pattern. In some cases, these techniques have shown the ability to provide further ridge detail, "filling in the gaps" of the developed mark. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) is presently the most advanced of the so-called 'surface analysis' techniques, in terms of compatibility with a number of fingermark enhancement processes and implementation in operational casework. However, for the use of this technique in major crimes to become widespread, compatibility with sequential processing must be demonstrated. This short study has assessed compatibility with a number fingermark processing sequences applied to natural marks on the adhesive side of brown (parcel) and clear tapes. Within the study undertaken, the results confirm the possibility to use MALDI MSI in sequence with multiple processes offering in some instances, complementary ridge detail with respect to that recovered from marks developed by conventional sequence processing.

Sample Treatment for Tissue Proteomics in Cancer, Toxicology, and Forensics.

Since the birth of proteomics science in the 1990, the number of applications and of sample preparation methods has grown exponentially, making a huge contribution to the knowledge in life science disciplines. Continuous improvements in the sample treatment strategies unlock and reveal the fine details of disease mechanisms, drug potency, and toxicity as well as enable new disciplines to be investigated such as forensic science.This chapter will cover the most recent developments in sample preparation strategies for tissue proteomics in three areas, namely, cancer, toxicology, and forensics, thus also demonstrating breath of application within the domain of health and well-being, pharmaceuticals, and secure societies.In particular, in the area of cancer (human tumor biomarkers), the most efficient and multi-informative proteomic strategies will be covered in relation to the subsequent application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA), due to their ability to provide molecular localization of tumor biomarkers albeit with different spatial resolution.With respect to toxicology, methodologies applied in toxicoproteomics will be illustrated with examples from its use in two important areas: the study of drug-induced liver injury (DILI) and studies of effects of chemical and environmental insults on skin, i.e., the effects of irritants, sensitizers, and ionizing radiation. Within this chapter, mainly tissue proteomics sample preparation methods for LC-MS/MS analysis will be discussed as (i) the use of LC-MS/MS is majorly represented in the research efforts of the bioanalytical community in this area and (ii) LC-MS/MS still is the gold standard for quantification studies.Finally, the use of proteomics will also be discussed in forensic science with respect to the information that can be recovered from blood and fingerprint evidence which are commonly encountered at the scene of the crime. The application of proteomic strategies for the analysis of blood and fingerprints is novel and proteomic preparation methods will be reported in relation to the subsequent use of mass spectrometry without any hyphenation. While generally yielding more information, hyphenated methods are often more laborious and time-consuming; since forensic investigations need quick turnaround, without compromising validity of the information, the prospect to develop methods for the application of quick forensic mass spectrometry techniques such as MALDI-MS (in imaging or profiling mode) is of great interest.

Investigation of infinite focus microscopy for the determination of the association of blood with fingermarks.

The determination of the type of deposition mechanism of blood within fingermarks at the scene of violent crimes is of great importance for the reconstruction of the bloodshed dynamics. However, to date, evaluation still relies on the subjective visual examination of experts. Practitioners encounter three types of scenarios in which blood may be found in fingermarks and they refer to the following three deposition mechanisms: (i) blood marks, originating from a bloodied fingertip; (ii) marks in blood, originating from a clean fingertip contacting a blood contaminated surface; (iii) coincidental deposition mechanisms, originating from a clean fingertip contacting a clean surface, leaving a latent fingermark, and subsequent contamination with blood. The authors hypothesised that, due to differences in distribution of blood in the furrows and on the ridges, the height of blood depositions on the ridges and furrows (and their relative proportions), will differ significantly across the three depositions mechanisms. A second hypothesis was made that the differences would be significant and consistent enough to exploit their measurement as a quantitative and objective way to differentiate the deposition mechanisms. In recent years, infinite focus microscopy (IFM) has been developed, allowing for the computational generation of a 3D image of the topology of a sample via acquisition of images on multiple focal planes. On these bases, it was finally hypothesised that the application of this technique would allow the distinction of deposition mechanisms (i) to (iii). A set of preliminary experiments were designed to test whether IFM was "fit for purpose" and, subsequently, to test if any of the three deposition mechanisms scenarios could be differentiated. Though IFM enabled the analysis of tape lifted samples with some success, for samples produced and analysed directly on the surface of deposition, the results show that the measurements from any scenario will be highly dependent on the original surface of deposition (both in terms of its nature and of the variable exposure to environment); as crime scenes exhibit a wide range of possible relevant surfaces of deposition, the technique showed to not have the desired wide appeal for inclusion into a standardised set of protocols within a routine crime scene workflow.

An update on MALDI mass spectrometry based technology for the analysis of fingermarks - stepping into operational deployment.

Since 2009, when Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) was firstly reported for the molecular mapping of latent fingermarks, the range of information and operational capabilities have steadily increased. Pioneering work from our Fingermark Research Group exploited different modalities, including Profiling (MALDI MSP), tandem mass spectrometry (MS/MS) and Ion Mobility MS/MS; a number of methodologies were also developed to conquer a main challenge, namely profiling the suspect and their actions prior to or whilst committing the crime. Suspect profiling here is no longer based on behavioural science but complements this discipline and the investigations by detecting and visualising the molecular make-up of fingermarks onto the identifying ridges. This forensic opportunity provides the link between the biometric information (ridge detail) and the corpus delicti or intelligence on the circumstances of the crime. In 2013, a review was published covering the research work and developments of four years supported by the Home Office, UK, and the local regional Police with some insights (and comparison) into similar research being reported employing other mass spectrometric techniques. The present review is an extensive update on the MALDI MS based methods' achievements, limitations and work in progress in fingermark analysis; it also offers an outlook on further necessary research into this subject. The main highlights are the increased number of possible information retrievable around a suspect and the more extended compatibility of this technology. The latter has allowed MALDI MS based methods to integrate well with current forensic fingerprinting, leading to the investigation of real police casework.

Implementation of MALDI MS profiling and imaging methods for the analysis of real crime scene fingermarks.

In the study presented here, four examples of crime scene fingermarks analysed by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI), in collaboration with the local Police Force, are reported. These marks are associated to high profile crimes such as drug dealing, murder and harassment. Following the application of forensic enhancement techniques by the CSI or the crime lab, one of the marks could be directly analysed on the surface of deposition and the others were analysed as primary lifts. In one case, no physical or molecular information was obtained whereas in two cases both ridge detail (albeit not useful for identification purposes) and molecular information could be retrieved. In one case, the intelligence gathered around the suspect's state of mind is, to date the most accomplished demonstration of the benefits and the operational feasibility MALDI MS based methods. These four casework examples are to be considered a first insight into the limitations, benefits and feasibility of MALDI MS based methods in the field; due to the extreme variability in the state of the mark, comprehensive evaluation of these aspects can only be undertaken upon the analysis of a large number of crime scene marks. However, this work does represent a significant advancement, compared to previous published work, as it demonstrates operational feasibility, with different levels of success, despite uncontrollable, unknown and unaccountable environmental and donor variability.

The Reed-Stanton press rig for the generation of reproducible fingermarks: Towards a standardised methodology for fingermark research.

In the search for better or new methods/techniques to visualise fingermarks or to analyse them exploiting their chemical content, fingermarks inter-variability may hinder the assessment of the method effectiveness. Variability is due to changes in the chemical composition of the fingermarks between different donors and within the same donor, as well as to differential contact time, pressure and angle. When validating a method or comparing it with existing ones, it is not always possible to account for this type of variability. One way to compensate for these issues is to employ, in the early stages of the method development, a device generating reproducible fingermarks. Here the authors present their take on such device, as well as quantitatively describing its performance and benefits against the manual production of marks. Finally a short application is illustrated for the use of this device, at the method developmental stages, in an emerging area of fingerprinting research concerning the retrieval of chemical intelligence from fingermarks.

A proteomic approach for the rapid, multi-informative and reliable identification of blood.

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence in the forensic investigation of serious offences. Because many of the current enhancement methods used by crime scene investigators are presumptive, the visualisation of blood is not always reliable nor does it bear additional information. In the work presented here, two methods employing a shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols employ both an in solution digestion method and a recently proposed procedure involving immobilization of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes). Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate.

Detection and mapping of illicit drugs and their metabolites in fingermarks by MALDI MS and compatibility with forensic techniques.

Despite the proven capabilities of Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) in laboratory settings, research is still needed to integrate this technique into current forensic fingerprinting practice. Optimised protocols enabling the compatible application of MALDI to developed fingermarks will allow additional intelligence to be gathered around a suspect's lifestyle and activities prior to the deposition of their fingermarks while committing a crime. The detection and mapping of illicit drugs and metabolites in latent fingermarks would provide intelligence that is beneficial for both police investigations and court cases. This study investigated MALDI MS detection and mapping capabilities for a large range of drugs of abuse and their metabolites in fingermarks; the detection and mapping of a mixture of these drugs in marks, with and without prior development with cyanoacrylate fuming or Vacuum Metal Deposition, was also examined. Our findings indicate the versatility of MALDI technology and its ability to retrieve chemical intelligence either by detecting the compounds investigated or by using their ion signals to reconstruct 2D maps of fingermark ridge details.

Direct detection of blood in fingermarks by MALDI MS profiling and imaging.

The determination of the presence of blood in fingermarks constitutes important intelligence in a criminal investigation as it helps to reconstruct the events that have taken place at a scene of crime. Various methodologies have been reported and are currently employed for the detection of the presence of blood including optical, spectroscopic and chemical development approaches. However, most methods only give an indication that blood may be present and, therefore, these methods are described as presumptive tests. Here we show the use of Matrix-Assisted Laser Desorption Ionisation Mass Spectrometry Profiling and Imaging (MALDI MSP and MALDI MSI) for the determination of the presence of blood in fingermarks by specifically detecting the molecules of haem and haemoglobin through their mass-to-charge ratios. Furthermore, preliminary experiments are shown which demonstrate that this technology is compatible with other methods currently employed for enhancing fingermarks in blood (or contaminated by blood). The application of the developed protocols to a crime scene blood trace, demonstrates the feasibility of using this technology in routine casework. These findings open up a new line of research for the development of robust MALDI MSP and MALDI MSI protocols for the detection and chemical imaging of bloodied marks.

Beyond the ridge pattern: multi-informative analysis of latent fingermarks by MALDI mass spectrometry.

After over a century, fingerprints are still one of the most powerful means of biometric identification. The conventional forensic workflow for suspect identification consists of (i) recovering latent marks from crime scenes using the appropriate enhancement technique and (ii) obtaining an image of the mark to compare either against known suspect prints and/or to search in a Fingerprint Database. The suspect is identified through matching the ridge pattern and local characteristics of the ridge pattern (minutiae). However successful, there are a number of scenarios in which this process may fail; they include the recovery of partial, distorted or smudged marks, poor quality of the image resulting from inadequacy of the enhancement technique applied, extensive scarring/abrasion of the fingertips or absence of suspect's fingerprint records in the database. In all of these instances it would be very desirable to have a technology able to provide additional information from a fingermark exploiting its endogenous and exogenous chemical content. This opportunity could potentially provide new investigative leads, especially when the fingermark comparison and match process fails. We have demonstrated that Matrix Assisted Laser Desorption Ionisation Mass Spectrometry and Mass Spectrometry Imaging (MALDI MSI) can provide multiple images of the same fingermark in one analysis simultaneous with additional intelligence. Here, a review on the pioneering use and development of MALDI MSI for the analysis of latent fingermarks is presented along with the latest achievements on the forensic intelligence retrievable.

Curcumin: a multipurpose matrix for MALDI mass spectrometry imaging applications.

Curcumin, 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene-3,5-dione, is a polyphenolic compound naturally present in the Curcuma longa plant, also known as tumeric. Used primarily as a coloring agent and additive in food, curcumin has also long been used for its therapeutic properties in a number of medical scenarios. Here, we report on an entirely novel use of curcumin; its extended structure of conjugated double bonds suggested the potential of this compound to be a good matrix assisted laser desorption ionization mass spectrometry (MALDI MS) matrix candidate. In the quest for novel and more efficient MALDI MS matrices, curcumin is revealed to be a versatile and multipurpose matrix. It has been applied successfully for the analysis of pharmaceuticals and drugs, for imaging lipids in skin and lung tissues, and for the analysis of a number of compound classes in fingermarks. In each case, the use of curcumin is shown to promote analyte ionization very efficiently as well as provide excellent mass spectral image quality.

Separation of overlapping fingermarks by matrix assisted laser desorption ionisation mass spectrometry imaging.

Latent fingermarks are impressions of the skin ridge pattern that are transferred by the accidental contact of fingertips with a deposition surface. The ability to enhance, lift and produce an image of a latent fingermark, for comparison and suspect match against a central fingerprint database, provides forensic investigators with what is still considered one of the most powerful means of biometric identification to date. Identification relies on the recovery, visualisation, extraction and comparison of local characteristics of the ridge pattern (minutiae) that are unique to individuals. Therefore, both for manual inspection of the minutiae and using automated ridge extraction algorithms, the clearer the ridge details, the more reliable and successful the match. Overlapping fingermarks pose a remarkable challenge in this context and are often encountered when developing marks from crime scenes. Here we propose the use of Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) to separate overlapping fingermarks using ion signals that are characteristic of each fingermark and that may be endogenous or exogenous in nature. In this work we show that the methodology works in a number of different scenarios both using manual inspection of the spectrum profile or a much quicker multivariate statistical analysis.

Isolation of enriched glial populations from post-mortem human CNS material by immuno-laser capture microdissection.

Isolating individual populations of cells from post-mortem (PM) central nervous system (CNS) tissue for transcriptomic analysis will provide important insights into the pathogenesis of neurodegenerative diseases. To date, research on individual CNS cell populations has been hindered by the availability of suitable PM material, unreliable sample preparation and difficulties obtaining individual cell populations. In this paper we report how rapid immunohistochemistry combined with laser capture microdissection (immuno-LCM) enables the isolation of specific cell populations from PM CNS tissue, thereby enabling the RNA profile of these individual cell types to be investigated. Specifically, we detail methods for isolating enriched glial populations (astrocytes, oligodendrocytes and microglia) and confirm this cell enrichment by polymerase chain reaction (PCR). In addition, the study details the numbers of each glial population required to obtain 50ng RNA, a suitable amount of starting material required to carry out microarray analysis that potentially may identify alterations of cell-specific genes and pathways associated with a range of neurodegenerative disorders.

Two-step matrix application for the enhancement and imaging of latent fingermarks.

Matrix deposition is a crucial aspect for successful matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) analysis. The search for more efficient protocols over the years has resulted in the devising of "dry matrix methods" in which the matrix is solely or preliminarily deposited as powder and acts in most cases as a seeding agent. Although not fully embraced by the MALDI MSI community, these methods have proven to be more efficient in terms of ion intensity, ion abundance, and ion images in the experimental circumstances they were employed. Here we report a novel two-step matrix application method, that we have named the "dry-wet" method, where the matrix is dusted onto the sample followed by solvent spray using a robotic device. The new method has been successfully applied to the detection and mapping of several analyte classes within latent fingermarks. Dusting the matrix generated the added advantage of enhancing the latent fingermarks which are invisible. This allows not only for an optical image to be taken of the fingermark in situ but also bridges the gap in the application of MALDI MSI technology in this field; with the use of the methodology reported, fingermark enhancement, recovery, and analysis from different surfaces is now compatible with subsequent MALDI MSI analysis thus allowing visual and chemical information to be obtained simultaneously.