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Syndromic approach for molecular diagnostics of infectious diseases
Infectious diseases, in particular lower respiratory infections and diarrhoea are among the top ten causes of death worldwide [1]. Many infectious diseases can be considered ‘syndromes’, i.e. a collection of symptoms that do not point to a specific causative agent. A ‘syndromic approach’ in diagnostics means to achieve, by a single test, a diagnosis without taking into account the symptoms themselves. In molecular diagnostics (MDx) of infectious diseases, this means the use of multiplex PCR panel assays to simultaneously detect genetic material from pathogens of different species and even different taxonomic levels, such as bacteria, viruses, parasites and fungi.
by Dr. Antoinette A. T. P. Brink and Dr. Guus F. M. Simons
Clinical syndromes and their causal pathogens
Respiratory infections
Respiratory infections are highly prevalent and the possible causative agents include several typical and atypical bacteria, as well as many viruses. Among the latter, the influenza virus particularly is associated with morbidity and mortality. As influenza A viruses can infect multiple hosts, including not only humans but also birds and swine, these viruses can undergo antigenic shifts that may result in pandemics such as the 2009 ‘Mexican flu’. Nowadays, many molecular diagnostics (MDx) tests are able to distinguish influenza A virus subtypes associated with non-human hosts, such as H5N7 avian flu. The presence of such a subtype in a patient with respiratory illness may be indicative for zoonosis, which increases the risk of a new pandemic. Besides influenza A virus, syndromic MDx panels generally detect influenza B virus, respiratory syncytial virus (RSV) A and B, adenovirus (AdV), human metapneumovirus (hMPV), parainfluenza virus (PIV) types 1–4 and human coronavirus (hCoV) types 229E, OC43, NL63 and HKU1, rhinovirus (RV) and enterovirus (EV). Bocavirus is not always included, although it is considered an important pathogen especially in children [2].
The merit of testing a broad respiratory panel is illustrated by a study conducted by the Dutch National Institute for Public Health and the Environment (RIVM) among community-dwelling elderly receiving annual influenza vaccination. Vaccine effectiveness was studied by determining the relative contribution of influenza and other respiratory pathogens to influenza-like illness (ILI), using an assay that detects 22 respiratory pathogens simultaneously (RespiFinder®) As expected, vaccination reduced the incidence of influenza, but the number of ILI episodes was similar between vaccinated and non-vaccinated individuals; non-Influenza viruses replaced influenza as a cause of ILI in vaccinated individuals [3].
This finding is in line with the recommendation of the American Society for Microbiology working group for respiratory virus testing not to restrict testing for specific respiratory viruses during certain seasons because with global travel, many ‘seasonal’ viruses can cause disease throughout the year [4]. Moreover, testing should not be restricted to certain patient populations, e.g. testing for RSV/hMPV only in children, because these viruses may cause severe disease in adults as well.
For fastidious bacteria causing atypical pneumonia, such as Legionella pneumophila, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis and B. parapertussis, MDx has largely replaced conventional culture.
For Streptococcus pneumoniae and other bacteria associated with typical pneumonia, PCR is not ready to replace conventional culture because these bacteria belong to the normal oral flora. Quantitative detection is necessary to distinguish colonization from infection, but sample quality criteria for this are lacking. Moreover, conventional culture remains necessary for antimicrobial susceptibility testing.
Gastroenteritis
Most gastroenteritis (GE) panels detect an extensive list of viruses, bacteria and parasites. It is self-evident that GE viruses such as norovirus, adenovirus types 40 and 41, and rotavirus are included. Although the clinical course of sapovirus and human astrovirus (HAstV) infections is generally milder than that of, for example, norovirus, they should be included in routine testing to identify outbreaks and to ensure proper care of patients at risk for severe complications, such as infants, elderly and immuno-compromised patients.
The parasites Cryptosporidium spp., Entamoeba histolytica and Giardia lamblia are included in all GE panels because their pathogenicity is well established. For Dientamoeba fragilis this is still a matter of debate, but it should be considered the causal factor of GE symptoms – and treated appropriately – after other causes have been excluded [5].
Regarding GE-causing bacteria, most commercially available tests detect the most common pathogenic Escherichia coli types (EHEC, ETEC, STEC, EPEC, and IEIC), Salmonella spp. and Yersinia enterocolitica. For Campylobacter it is important to detect all pathogenic species, i.e. C. jejuni, C. coli, C. upsaliensis and C. lari [6], the latter two of which are not included in some commercially available assays.
In contrast, for Vibrio cholerae it is important to distinguish the only two serotypes that can cause outbreaks and infections should be treated actively, to avoid overtreatment.
Additional pathogens may be included, but test results may be difficult to interpret owing to (i) frequent contamination from the environment or reagents (e.g. Aeromonas spp.) or (ii) conflicting data regarding pathogenicity (e.g. Plesiomonas shigelloides).
Infections of the central nervous system
The differential diagnosis for patients with suspected meningitis/encephalitis (ME) includes infectious as well as non-infectious causes that cannot be distinguished on the basis of symptoms. A recent study showed that in the majority of patients suspected of a central nervous system (CNS) infection, the etiology could not be found even with the most comprehensive commercially available MDx tests [7]. In line with this, the use of a diagnostic stewardship approach is advocated, including cerebrospinal fluid white blood cell count to prevent unnecessary use of expensive tests [8]. Having said that, true CNS infections are medical emergencies that require rapid pathogen identification to allow timely and appropriate clinical intervention.
Causative agents of CNS infections include enteroviruses, parechoviruses and all eight members of the human herpes virus family, the most prevalent of which are herpes simplex virus types 1 and 2. The other herpes viruses, such as cytomegalovirus, varicella-zoster virus, human herpes virus 6, and Epstein-Barr virus, are mostly seen in immunocompromised patients such as transplant recipients [9].
Measles and mumps viruses may also cause CNS infections, especially in regions where vaccine coverage is sub-optimal due to, for example, a weak healthcare system or vaccine refusal. Of note, when this paper was drafted, four countries in the European Union (Albania, Czechia, Greece and the United Kingdom) had recently lost their measles elimination status previously assigned by the World Health Organization.
Meningitis-causing bacteria usually belong to the normal oropharyngeal flora, and may enter the CNS by anatomic defects in the natural barriers, or defects in the immune system. Streptococcus pneumoniae is the most common cause of community-acquired meningitis in non-neonates worldwide, followed by Neisseria meningitidis. Other bacterial pathogens in meningitis are Listeria monocytogenes, Haemophilus influenzae, Staphylococcus aureus and Borrelia burgdorferi.
Vaccination campaigns against various serogroups of these bacteria are ongoing. As serogroup replacement occurs as a consequence of vaccination, it is important that in vitro diagnostic kits detect all serogroups, and that assay designs are checked periodically for this.
Sexually transmitted infections
Various bacteria, parasites and viruses can cause sexually transmitted infections (STI).
STIs can be non-symptomatic, but if left untreated they can have severe sequelae including permanent infertility or ectopic pregnancy.
STI test panels should at least include Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis. For a syndromic approach, herpes simplex virus types 1 and 2 and Treponema pallidum should be included.
The editorial board of the European STI Guidelines recommends not to test routinely for Mycoplasma hominis and Ureaplasma parvum. In addition, in men with symptomatic urethritis, Ureaplasma urealyticum should only be treated after C. trachomatis, N. gonorrhoeae and M. genitalium have been excluded [10].
Several subtypes of STI require non-standard therapy. For example, the ‘L’ serovars of C. trachomatis can cause lymphogranuloma venereum (LGV) and proctitis, which should be treated differently from other C. trachomatis infections [11].
Furthermore, N. gonorrhoeae strains have emerged that are resistant to all antimicrobials used for treatment, owing to point mutations and/or genetic recombination with commensal Neisseria species [12]. In addition, the change from doxycycline to azithromycin as the first-line treatment for C. trachomatis and non-gonococcal urethritis has resulted in selection of macrolide-resistant M. genitalium strains [13]. Fortunately, such pathogen properties can be distinguished by molecular methods and, in fact, several commercially available multiplex MDx assays can do this already.
MDx methods for syndromic approach
Laboratory-developed tests
Laboratory-developed PCR assays for infectious diseases have been in use since the early 1990s [14].
Generally, laboratory-developed tests (LDTs) run on conventional 96-well realtime PCR systems, allowing the testing of large batches of samples. Most LDTs are TaqMan-based, generally limiting multiplexing to four targets per reaction. A syndromic approach is, therefore, only possible by running several reactions (up to eight to cover a full respiratory panel) per sample, which limits throughput.
Although the cost-of-goods for LDTs is low, testing can be laborious and time-consuming and requires highly skilled laboratory personnel.
Commercial: medium to high throughput
Clinical laboratories needing a syndromic approach and medium to high throughput may use commercial assays instead of LDT. To exceed the multiplexing capacity of LDTs, manufacturers have developed proprietary methods. For example, Luminex’s platform uses fluorescently labelled bead array technology with dedicated instruments. Korean Seegene’s assays include melting curve analysis or differential detection temperatures to allow distinction of multiple targets per fluorescent channel.
Commercial: random access/low throughput
Examples of commercial random access systems are the FilmArray by bioMérieux, the QIAstat-Dx by QIAGEN and the ePlex by GenMark. The high price for the dedicated instruments and high price-per-test can be an argument against implementation in routine use if large amounts of samples are processed. Hence, these systems are particularly suitable for point-of-care purposes or when laboratory skills of personnel are limited. However, the ease-of-use may conceal to inexperienced users that these tests are actually very sensitive, and careful reaction set-up and cleanliness of the environment are needed to avoid false positives. It is self-evident that (unexpected) positive results should be interpreted in the context of clinical symptoms. Moreover, the ISO 15189 standard for medical laboratories recommends users to run additional control materials from independent third parties.
Summary
A syndrome-based approach using broad panel MDx assays may assist in timely diagnosis of respiratory infections, GE, CNS infections and STI.
Syndrome-based MDx results in a decrease in the number of chest radiographs, reduced admission rates, fewer barrier nursing days [15], shorter duration of hospitalization, more appropriate prescription of antivirals, better antibiotic stewardship and decreased duration of antimicrobial use [16–18], which is likely to result in less antibiotic resistance in the long term.
In addition, broad panel tests provide useful information about epidemiology, seasonality and possibly clinically relevant co-infections
Figure 1: Example of high multiplexing (the 2SMARTFinder® principle of Dutch firm PathoFinder)
Schematic representation of the PathoFinder 2SMARTFinder® test principle: (a) Step 1 (pre-amplification): specific multiplex target enrichment. (b) Step 2 (2SMART reaction): signal amplification by means of 2SMART primers each consisting of a targetspecific sequence (yellow/blue) and a universal sequence (grey/green). Each reverse 2SMART primer contains a stuffer/barcode sequence (red) for detection. The combined action of the 2SMART primers and the universal primers, of which the reverse is labelled with fluorescein amidites (FAM), results in the generation of (c) a FAM-labelled PCR product. (d) The reaction mixture contains labelled SMART probes complementary to each stuffer. (e) When a probe hybridizes to its corresponding stuffer in the reaction product, the FAM label acts as a Förster Resonance Energy Transfer (FRET) donor and the label in the SMART probe as an acceptor, resulting in the emission of light that can be measured in real-time. Finally, the temperature is increased, resulting in dissociation of the probe-stuffer hybrid and a sharp decline in fluorescence (f). The negative derivative of this graph shows the actual melting peaks (g). The stuffer/probe sequence determines the position of the melting peak and reveals which pathogen was present in the sample. −Δ(F)/dT, negative derivative of the change in fluorescence (F) as a function of temperature (T).
Figure 2: Typical result read-out on LightCycler 480
Example of the read-out of the RespiFinder® 2SMART assay mix 1 in the carboxyrhodamine (ROX™) channel of a LightCycler 480 II real-time PCR instrument. AdV, adenovirus; hMPV, human metapneumovirus; Inf A, influenza A virus; Inf B, influenza B virus; RSVA, respiratory syncytial virus A; RSVA, respiratory syncytial virus B
References
1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. The Lancet 2012; 380: 2095–2128.
2. Ma X, Conrad T, Alchikh M, Reiche J, Schweiger B, Rath B. Can we distinguish respiratory viral infections based on clinical features? A prospective pediatric cohort compared to systematic literature review. Rev Med Virol 2018; 28: e1997.
3. van Beek J, Veenhoven RH, Bruin JP, van Boxtel RAJ, de Lange MMA, Meijer A, Sanders EAM, Rots NY, Luytjes W. Influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections. J Infect Dis 2017; 216: 415–424.
4. Ginocchio CC, McAdam AJ. Current best practices for respiratory virus testing. J Clin Microbiol 2011; 49: S44–S48.
5. Van Gestel RSFE, Kusters JG, Monkelbaan JF. A clinical guideline on Dientamoeba fragilis infections. Parasitology 2018; 1–9.
6. Klena JD, Parker CT, Knibb K, Ibbitt JC, Devane PML, Horn ST, Miller WG, Konkel ME. Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA. J Clin Microbiol 2004; 42: 5549–5557.
7. Sall O, Thulin Hedberg S, Neander M, Tiwari S, Dornon L, Bom R, Lagerqvist N, Sundqvist M, Molling P. Etiology of central nervous system infections in a rural area of Nepal using molecular approaches. Am J Trop Med Hyg 2019; 101(1): 253–259.
8. Messacar K, Robinson CC, Dominguez SR. Letter to the editor: economic analysis lacks external validity to support universal syndromic testing for suspected meningitis/encephalitis. Future Microbiol 2018; 13: 1553–1554.
9. Chadwick DR. Viral meningitis. Br Med Bull 2005; 75–76: 1–14.
10. Horner P, Donders G, Cusini M, Gomberg M, Jensen JS, Unemo M. Should we be testing for urogenital Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum in men and women? – a position statement from the European STI Guidelines Editorial Board. J Eur Acad Dermatol Venereol 2018; 32: 1845–1851.
11. Ceovic R, Gulin SJ. Lymphogranuloma venereum: diagnostic and treatment challenges. Infect Drug Resist 2015; 8: 39–47.
12. Ameyama S, Onodera S, Takahata M, Minami S, Maki N, Endo K, Goto H, Suzuki H, Oishi Y. Mosaic-like structure of penicillin-binding protein 2 gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime. Antimicrob Agents Chemother 2002; 46: 3744–3749.
13. Jensen JS. Mycoplasma genitalium: yet another challenging STI. Lancet Infect Dis 2017; 17: 795–796.
14. Claas HC, Wagenvoort JH, Niesters HG, Tio TT, Van Rijsoort-Vos JH, Quint WG. Diagnostic value of the polymerase chain reaction for Chlamydia detection as determined in a follow-up study. J Clin Microbiol 1991; 29: 42–45.
15. Goldenberg SD, Bacelar M, Brazier P, Bisnauthsing K, Edgeworth JD. A cost benefit analysis of the Luminex xTAG Gastrointestinal Pathogen Panel for detection of infectious gastroenteritis in hospitalised patients. J Infect 2015; 70: 504–511.
16. Rappo U, Schuetz AN, Jenkins SG, Calfee DP, Walsh TJ, Wells MT, Hollenberg JP, Glesby MJ. Impact of early detection of respiratory viruses by multiplex PCR assay on clinical outcomes in adult patients. J Clin Microbiol 2016; 54: 2096–2103.
17. Andrews D, Chetty Y, Cooper BS, Virk M, Glass SK, Letters A, Kelly PA, Sudhanva M, Jeyaratnam D. Multiplex PCR point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use. BMC Infect Dis 2017; 17: 671–671.
18. Echavarria M, Marcone DN, Querci M, Seoane A, Ypas M, Videla C, O’Farrell C, Vidaurreta S, Ekstrom J, Carballal G. Clinical impact of rapid molecular detection of respiratory pathogens in patients with acute respiratory infection. J Clin Virol 2018; 108: 90–95.
The authors
Antoinette A.T.P. Brink* PhD, Guus F.M.
Simons PhD PathoFinder B.V., 6229 EG Maastricht,
The Netherlands
*Corresponding author
E-mail: antoinette.brink@pathofinder.com
Increased sensitivity of detecting and monitoring plasma cell disorders using MS
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
1. Willrich MAV, et al. Clin Biochem 2018; 51: 38–47.
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3. Katzmann JA, et al. Clin Chem 2009; 55(8): 1517–1522.
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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
