Standard drug testing is regularly carried out using urine, blood or oral fluid. However, fingerprints present a good alternative, as the sample collection is non-invasive, rapid and safe. Herein, we describe the application of two different testing methods for the detection of cocaine in fingerprint samples.

by Dr Catia Costa, Dr Mahado Ismail and Dr Melanie J. Bailey

Drug abuse in the United Kingdom is on the rise and it is a cause for concern, with widespread financial and social implications [1, 2]. The ever-growing drug and alcohol culture in the UK has led to the implementation of workplace drug testing in many industries, especially those in high-risk operational environments. Consequently, there has been a surge in the demand for drug-screening suppliers to develop faster and more reliable testing. This demand is set to increase the market value of drug and alcohol testing in the UK from £167 million to £231 million by 2019 [3].

Conventionally, drug testing is carried out using biological matrices such as blood, urine and, more recently, oral fluid. These matrices and methods of analysis, although established, present a few problems relating to sample collection and transportation. The collection of blood requires medically trained personnel and sample collection is considered invasive, whereas urine carries privacy concerns. Oral fluid is an alternative matrix used for non-invasive drug testing, although sample collection can be time-consuming. All these three matrices are also biohazardous, making sample storage and transportation a potential issue. The potential use of fingerprints for drug testing has become the subject of many recent publications. Fingerprint samples present a good alternative for drug testing as collection is non-invasive and rapid, and there are no known biohazards associated with the sample. Additionally, the fingerprint pattern can be used for donor identification.

Chemical analysis of fingerprints
The chemical information embedded in a fingerprint sample has been reviewed by many, and several publications have explored the detection of substances such as cocaine [4–6], heroin [7], methadone [8], lorazepam [9], methamphetamines [10], caffeine [11] and cough medicine [12] in fingerprints after administration of the substances. These reports are predominantly based on liquid chromatography-mass spectrometry (LC-MS), which is very well established in the field of toxicology for its quantitative potential as well as its sensitivity and reliability. New advances in the field of mass spectrometry saw the rise of ambient ionization mass spectrometry techniques that allow the sample to be analysed in its native state, under ambient conditions. Examples include desorption electrospray ionization (DESI), liquid extraction surface analysis (LESA) and paper spray, which have been applied to the detection of cocaine and metabolites in fingerprint samples [4–6].

Most of these reports in the literature have looked at fingerprint samples collected after administration of the substances. However, no research has investigated the significance of the detection of these substances compared to a large background population of non-drug users. This is of particular importance as a positive test result may be the outcome of contamination by contact with contaminated surfaces or handling the parent drug rather than ingestion. This directly highlights the need for a sampling strategy that removes any contact residue while providing enough fingerprint material for the analysis.

Detection of cocaine in fingerprints
The detection of cocaine in fingerprints has been studied and reported by Ismail et al. [7]. This study looked at fingerprints collected from the background population (i.e. non-drug users) and from patients at a drug rehabilitation clinic. Both sets of samples were collected as presented and after handwashing, followed by wearing nitrile gloves for 10 minutes. Fingerprint results were supported by oral fluid analysis and patient testimony. Analysis of samples collected from patients (n=13) at the rehabilitation clinic yielded a 100% detection rate for cocaine for samples collected as presented and after handwashing. However, the detection of the cocaine metabolite, benzoylecgonine (BZE), decreased from 94% from samples collected as presented, to 87% for samples collected after handwashing. To evaluate the significance of the results above, fingerprint samples collected from the background population were analysed to investigate the prevalence of these substances in non-drug users. Samples collected as presented (n=99 samples) returned a 13% and 5% detection rate for cocaine and BZE, respectively. After handwashing, cocaine was only detected in 1% of the samples analysed (n=100) and no BZE was present. These findings suggest that cocaine can be detected in the background population owing to environmental exposure (e.g. contact with bank notes). However, after using a handwashing procedure, cocaine and benzoylecgonine were not prevalent. Collection of fingerprint samples after a hand-cleaning procedure is therefore advantageous to reduce potential false-positive rates that can be observed from environmental exposure.

As previously mentioned, the use of chromatographic methods is well established in the field of toxicology. However, such methods often rely on extensive sample preparation and analysis. To overcome this issue we have developed paper spray-mass spectrometry (PS-MS) for the detection of cocaine in under 4 minutes from fingerprints collected from patients seeking treatment at a rehabilitation centre [5]. For this method fingerprints are collected on a triangular piece of paper, which is in turn placed on the paper spray source for analysis. An internal standard, solvent and voltage are applied to the paper, resulting in the extraction and ionization of the fingerprint residues before detection on the mass spectrometer (Fig. 1). The method was evaluated with 239 fingerprint samples collected from drug users at the National Health Service (NHS)  rehabilitation clinics and from the background population. A positive result was based on the detection of cocaine or one of its two main metabolites, BZE and ecgonine methyl ester (EME). A 99% true-positive rate was achieved on the samples collected from patients at drug rehabilitation centres, which was supported by standard saliva drug testing and patient testimony. Analysis of samples collected from the general population yielded a 2.5% false-positive rate. This follows from the work by Ismail et al. [7] described above, where in the absence of a hand-cleaning procedure cocaine was detected in the background population. Both studies highlight the need for a well-defined sample collection procedure to eliminate false-positive results while maintaining true-positives.

This method has since its publication been shortened to 30 seconds and it has also been applied to the detection of heroin, morphine, codeine, 6-AM and explosive materials. This highlights the potential for the technique to be on a par with current testing methods that target a wide range of substances.

Fingerprint visualization
Another advantage of using fingerprints for drug testing is the possibility to integrate a fingerprint visualization step for donor identification. This would be of particular benefit for preventing cheating and also in cases of disputed results where one would be able to prove that the results were derived from the correct person. Silver nitrate was used to visualize fingerprint samples collected from drug users by treating the substrate before sample collection. Upon collection, samples were exposed to ultraviolet light to bring out the fingerprint pattern (Fig. 2). Analysis of fingerprint samples collected from drug users after silver nitrate development yielded a 100% detection rate for cocaine, showing great potential for this development step to be included in the fingerprint testing routine.

The future: treatment adherence monitoring
Treatment non-adherence is a well-known problem in the NHS and it is estimated that it can cost over £500 million each year [13]. Thus, the establishment of an adherence monitoring tool could result in substantial savings for the NHS. Fingerprint testing offers the opportunity for remote testing where the samples can be collected by the patient at home and sent to the laboratory for analysis. In cases of non-adherence, medical professionals may intervene and ensure the patient is receiving adequate treatment. This is of particular interest for conditions known to have poor adherence rates such as diabetes, cardiovascular diseases and mental health disorders [14] or for highly infectious diseases such as tuberculosis.

References
1. Barber S, Harker R, Pratt A. Human and financial costs of drug addiction. House of Commons Library 2017.
2. Health matters: preventing drug misuse deaths (GOV.CO.UK2017). Public Health England 2017 (https: //www.gov.uk/government/publications/health-matters-preventing-drug-misuse-deaths/health-matters-preventing-drug-misuse-deaths).
3. Eurofins Workplace Drug Testing launches new holistic ‘wrap around service’ to assist UK plc. Eurofins 2018 (https: //www.eurofins.co.uk/forensic-services/press-releases/uk-growing-drug-culture/).
4. Bailey MJ, Bradshaw R, Francese S, Salter TL, Costa C, Ismail M, Webb RP, Bosman I, Wolff K, de Puit M. Rapid detection of cocaine, benzoylecgonine and methylecgonine in fingerprints using surface mass spectrometry. Analyst 2015; 140(18): 6254–629.
5. Costa C, Webb R, Palitsin V, Ismail M, de Puit M, Atkinson S, Bailey MJ. Rapid, secure drug testing using fingerprint development and paper spray mass spectrometry. Clin Chem 2017; 63(11): 1745–17525.
6. Bailey MJ, Randall EC, Costa C, Salter TL, Race AM, de Puit M, Koeberg M, Baumert M, Bunch J. Analysis of urine, oral fluid and fingerprints by liquid extraction surface analysis coupled to high resolution MS and MS/MS – opportunities for forensic and biomedical science. Anal Methods 2016; 8(16): 3373–3382.
7. Ismail M, Stevenson D, Costa C, Webb R, de Puit M, Bailey M. Noninvasive detection of cocaine and heroin use with single fingerprints: determination of an environmental cutoff. Clin Chem 2018; 64(6): 909–917.
8. Jacob S, Jickells S, Wolff K, Smith N. Drug testing by chemical analysis of fingerprint deposits from methadone-maintained opioid dependent patients using UPLC-MS/MS. Drug Metab Lett 2008; 2(4): 245–247.
9. Goucher E, Kicman A, Smith N, Jickells S. The detection and quantification of lorazepam and its 3-O-glucuronide in fingerprint deposits by LC-MS/MS. J Sep Sci 2009; 32(13): 2266–2272.
10. Zhang T, Chen X, Yang R, Xu Y. Detection of methamphetamine and its main metabolite in fingermarks by liquid chromatography-mass spectrometry. Forensic Sci Int 2015; 248: 10–14.
11. Kuwayama K, Tsujikawa K, Miyaguchi H, Kanamori T, Iwata YT, Inoue H. Time-course measurements of caffeine and its metabolites extracted from fingertips after coffee intake: a preliminary study for the detection of drugs from fingerprints. Anal Bioanal Chem 2013; 405(12): 3945–3952.
12. Kuwayama K, Yamamuro T, Tsujikawa K, Miyaguchi H, Kanamori T, Iwata YT, Inoue H. Time-course measurements of drugs and metabolites transferred from fingertips after drug administration: usefulness of fingerprints for drug testing. Forensic Toxicol 2014: 32(2): 235–242.
13. Trueman P, Taylor D, Lowson K, Bligh A, Meszaros A, Wright D, Glanville J, Newbould J, Bury M, et al. Evaluation of the scale, causes and costs of waste medicines. York Health Economics Consortium/School of Pharmacy, University of London 2010.
14. Cutler RL, Fernandez-Llimos F, Frommer M, Benrimoj C, Garcia-Cardenas V. Economic impact of medication non-adherence by disease groups: a systematic review. BMJ Open 2018; 8(1): e016982.

The authors
Catia Costa*¹ PhD, Mahado Ismail² PhD and Melanie J. Bailey² PhD
¹Ion Beam Centre, University of Surrey, Surrey, GU2 7XH, UK
²Department of Chemistry, University of Surrey, Surrey, GU2 7XH, UK

*Corresponding author
E-mail: c.d.costa@surrey.ac.uk

Plasma cell disorders are detected in the clinical lab by finding the monoclonal immunoglobulin (M-protein) they produce. Serum protein electrophoresis methods have been employed widely to detect and isotype M-proteins. Increasing demands to detect residual disease and new therapeutic monoclonal immunoglobulin treatments have stretched electrophoretic methods to their limits. Newer techniques based on mass spectrometry are emerging which have improved clinical and analytical performance. These techniques are beginning to gain traction within routine clinical lab testing.

by Dr David L. Murray

Background
In a healthy immune system, the terminally differentiated white blood B-cells (i.e. plasma cells) each produce a unique immunoglobulin (Ig, or antibody) which was selected for by its fitness to bind to foreign invaders (antigens). This legion of plasma cells resides within our bone marrow and serves as a protective library manufacturing a diverse protective cacophony of Ig proteins whose aim is to protect us from recurrent infections. The total production of Igs in a healthy individual is remarkably highly regulated in the non-infected state with no particular plasma cell out-producing other plasma cells. As a result, the electrophoretic separation of healthy human serum results in a cathodically broad distribution of Ig proteins, which is labelled the gamma region (Fig. 1a).

In contrast, plasma cell proliferative disorders (PCDs) consist of a group of diseases stemming from clonal proliferation of a dysregulated plasma cell clone. PCDs range from relatively common benign conditions, such as monoclonal gammopathy of undetermined significance (MGUS), to frank malignant conditions, such as multiple myeloma (MM) [1]. Central to the detection of PCDs in serum is the detection of the over-produced monoclonal Ig by the dysregulated plasma clone (termed M‑protein or paraprotein). M‑proteins are a relatively common laboratory finding occurring in approximately 3 % of adults over the age of 50 [2]. The majority of these patients will live unaffected by the presence of the M‑protein while some patients will progress to more serious disease, such as MM, at a rate of 1 % per year. Currently, it is not possible to know which patient is going to progress and patients with an M‑protein undergo surveillance for M‑protein concentration changes yearly.

Electrophoresis-based assays
By nature, M‑proteins are heterogeneous and thus diverse methodologies are currently used to detect, characterize and quantitate serum M‑proteins in the clinical laboratory. Serum protein electrophoresis (PEL) was the first method available to detect and quantitate M‑proteins. To increase the specificity and sensitivity, a second technique known as immunofixation electrophoresis (IFE) enables establishment of M‑protein isotype (IgG, IgA, IgM, IgD, IgE or free light chain kappa or lambda) by examining multiple electrophoretic gel lanes in which the serum proteins were ‘fixed’ to the gel using reagents specific for human immunoglobulin components
(Fig. 1). A third assay, the serum free light chain (sFLC) assay, uses specific antibodies for quantitation of circulating free kappa and lambda light chains. This assay has demonstrated superior detection of PCDs, such as amyloid light chain (AL) amyloidosis, which can result from low levels of circulating monoclonal free light chains [3]. Currently, the International Myeloma Working Group recommends a panel of serum tests that include PEL, IFE and a sFLC assay quantitation to maximize the sensitivity of PCD screening [4].

Need for improved detection sensitivity
At our institution, agarose gel electrophoresis methods (PEL and IFE) have been used for detecting M‑proteins since 1967. While the utility of the electrophoretic methods to screen and monitor PCDs has been well established, several changes in the treatment of PCDs are pushing these methods to their analytical limits. Dramatic improvement in the treatment response of MM patients to new chemotherapies and immunotherapies is challenging long-held assumptions about this ominous disease. In particular, there is renewed hope that MM may be curable and perhaps it is time to start treating MM patients until all signs of the disease are eradicated. The long-standing routine serum electrophoretic methods are not capable of providing the analytical sensitivity needed to assess minimal residual disease (MRD). A few laboratorians have turned to using bone marrow biopsies to hunt for traces of the malignant plasma cells by high sensitivity flow cytometry and next-generation sequencing [5, 6]. In addition, new monoclonal therapeutic antibodies (t‑mAbs) designed to eradicate malignant plasma cells are producing interferences making it difficult to distinguish between a patient’s M‑protein and the t‑mAb drug. A search for a more convenient serum-based test to complement bone marrow MRD detection and aid in resolving t‑mAb interferences was sought to address limitations in traditional testing. Mass spectrometry (MS) is aptly suited for this task as the improvements in MS instrumentation and techniques have resulted in increased resolution and mass accuracy that have outpaced improvements in electrophoresis.
MS-based methodsFor Igs, both the overall charge of the protein (the basis of electrophoretic separation) and the mass of the protein (the basis of MS separation) are diverse among Igs owing to Ig gene rearrangement in which the adaptive immune system optimizes the affinity of the antigen binding region of the Ig to its target antigen. The unique amino acid sequence of the antigen binding domain results in a unique molecular mass (and peptide sequence) which is the basis of the mass spectrometric detection. Efforts to optimize M‑protein detection by MS have resulted in two methods differing in the analytical target used to detect the M‑protein. One method based on a tryptic digest of Igs and using selective reaction monitoring (SRM) MS to detect unique peptides from the Ig antigen binding region (also termed the ‘clonotypic’ peptide approach) [7] and a second method based on disassembling Igs by chemical reduction and measuring the mass distribution of Ig light chain [termed monoclonal immunoglobulin Rapid Accurate Mass Measurement (miRAMM)] [8]. Of these two approaches, the miRAMM method was suitable for adaptation to our high volume reference laboratory. The adaptation of the miRAMM method to MALDI-TOF mass spectrometers [9] eliminated the need for chromatography and allowed for throughputs suitable for PCD screening. The simplicity of MALDI-TOF data files also allowed our lab to build software capable of rapidly displaying multiple spectra which can be automatically analysed for an M‑protein. The current clinically validated version of the assay consists of five separate immune-enrichments for IgG, IgA, IgM, kappa and lambda which are separately analysed and the light chain mass distributions are examined for a ‘spike’ in a similar fashion to gel electrophoretic densitometry (Mass-Fix; Fig. 2). Mass-Fix has demonstrated overall superior analytical and clinical sensitivity to serum IFE [9, 10]. Mass-Fix has been automated and validated as a laboratory developed test and our one-year experience has confirmed that the assay is robust, sensitive and more labour efficient than our traditional gel IFE assay.
One of the benefits of using Mas-Fix over electrophoresis is the ability to determine a fundamental feature of the M‑protein, its light chain mass. Reporting the light chain mass allows for a more specific description of M‑protein than is currently available by electrophoresis. Current reporting of serum electrophoresis allows for placing an M‑protein within a region of the electropherorgram (alpha, beta or gamma) which is less specific than reporting an IgG kappa M‑protein with a light chain mass of 23 425 Da. Using the mass of the M‑protein light chain could allow other clinical labs using MS to assess the same patient for over-expressed clones of the same light chain mass increasing the confidence of M‑protein identity. By measuring the mass of the light chain of a t‑mAb, the lab will be able to determining if the detected over-expressed clone is due to the presence of a t‑mAb (such as daratumumab) or the patient’s M‑protein [11]. Additionally, the mass of the M‑protein light chain detected in other body fluids, such as urine, was found to be the same as in serum. This again affords more specificity than is currently available by electrophoresis.

The Mass-Fix assay has also shed light on M‑protein structural features that were not previously appreciated using electrophoretic techniques. In particular, the presence of monoclonal Ig light chains with masses outside the expected mass range were encountered in a small subset of patients. These light chains also had broader mass ranges than typically encountered with M‑proteins. Additional work revealed these light chains contained N-linked glycosylation [12]. Furthermore, patients with light chain glycosylated M‑proteins were found to be more likely to have a rarer form of a PCD (AL amyloidosis) than patients without light chain glycosylation.

Challenges and future perspectives
Challenges remain for these new assays to gain broad acceptance in the medical field. One feature that facilitates acceptance is Conformité Européene (CE) or U.S. Food and Drug Administration (FDA) approval in a format that is scalable and generalizable to a majority of clinical labs. Electrophoretic methods were employed prior to the FDA 510K process and thus have been grandfathered into the FDA approval system. This will not be the case for newer MS assays and thus time will be needed to get FDA approval. With increasing sensitivity, hematologists have also expressed concern over the potential increase in the detection of pre-malignant benign condition MGUS, as this would increase the number of consults.
These challenges need to be assessed in light of the numerous clinical advantages. The addition of the mass measurement allows for simpler conformation of peak as to its origin: disease or t‑mAb, the discovery of new risk factors for the formation of AL amyloidosis, and the ability to standard the detection from lab to lab.

Figure 1. Traditional detection of M-protein by immunofixation electrophoresis. (a) Healthy human serum demonstrating the albumin, alpha 1, alpha 2, beta and the broad gamma region which results from the diverse repertoire of Igs with slightly differing amino acid sequences and hence overall charge. (b) A patient with a plasma cell disorder demonstrating a relatively restricted band in the gamma region with immunofixation with anti-IgG (G) and anti (K) consistent with an IgG kappa M-protein.
Figure 2. Comparison of traditional immunofixation results and the new Mass-Fix spectra. (a) Healthy human serum demonstrating broad gamma region of IFE (left) and normal Gaussian [LC+2] m/z distribution for all immune-enrichments (IgG (black top), IgA (black middle), IgM (black, lower), kappa (orange, all spectra) and lambda (blue, all spectra). (b) A patient with a plasma cell disorder demonstrating a relative restricted band in the gamma region consistent with IgG kappa (left) and a non-Gaussian distribution of light chains with a peak in the IgG light mass distribution (black top) along with same peak in the total kappa light chain mass distribution (orange).

References
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The author
David L. Murray MD, PhD
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55906, USA
E-mail: Murray.David@mayo.edu