Metastatic breast cancer is a highly heterogeneous, rapidly evolving and devastating disease that challenges our ability to find curative therapies. RAS pathway activation is an understudied research area in breast cancer. EGFR/RAS pathway activation is prevalent in breast cancer with poor prognosis. The prognostic RAS pathway biomarkers can be used to identify resistant tumour clones, stratify patients and guide therapies.

by L. L. Siewertsz van Reesema, M. P. Lee, Dr V. Zheleva, Dr J. S. Winston,
Dr C. F. O’Connor, Prof. R. R. Perry, Prof. R. A. Hoefer and Prof. A. H. Tang

Introduction
Breast cancer currently represents the second leading cause of cancer-related deaths among women in the United States [1]. In 2016 alone, an estimated 246 660 women will be diagnosed and an additional 40 450 women are expected to succumb to the disease process [1, 2]. By 2030, there are projected to be 294 000 new cases, making breast cancer a growing public health concern [3–5]. The increased breast cancer screenings, major technical advancements in breast cancer imaging and early detection, and targeted anti-cancer therapies have contributed to significant decreases in morbidity and mortality since the 1970s, and now more than 90% of patients with early-stage breast cancer have expected survival of much more than 5 years [2, 5]. Despite these amazing advancements, the prognosis for patients with high-grade, locally advanced and metastatic breast cancers remains poor with average survival less than 2 years [2, 6–8]. State-of-the-art treatments, such as anti-HER2 therapy, anti-ER therapy, anti-PI3K and anti-mTOR therapy, tumour genome-guided combination therapies, stem cell therapy, and anti-CTLA-4/PD1 immunotherapy, alone or in combination, are not curative in eradicating metastatic breast cancer [9–14]. The clinical reality is that despite similar clinical diagnoses and presentation, patients often display diverse tumour response to standard therapies. The intrinsic diversity and evolving heterogeneity of their mammary tumours can become more pronounced in relapsed or metastatic tumours after therapeutic intervention and clonal selection by ineffective treatment. This de novo and acquired tumour heterogeneity leads to diverse tumour responses in neoadjuvant and adjuvant setting following standard-of-care therapies, which in turn leads to varied clinical outcome and disparity in patient survival [6, 15, 16].

Currently, there are no reliable clinical biomarkers that can be used to consistently predict survival of patients with late-stage or metastatic breast cancers [6]. Classically utilized breast cancer biomarkers, such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) molecular subtypes, do not correlate with survival outcomes nor do they predict tumour responses to guided therapies in invasive disease [6]. Patients with malignant tumours are often subjected to full regimens of toxic therapies followed by a period of uncertainty awaiting to learn the response of their tumours and efficacy of their treatments. Unfortunately, some patients may be over-treated, which compromises their long-term quality of life, or, conversely, some patients may be under- or incorrectly treated and thus miss a critical window of opportunity to change prescribed anticancer regimens if therapy-resistant tumours persist after aggressive therapy. There is a pressing need to identify reliable, sensitive, and robust prognostic biomarkers to closely monitor tumour response in real-time, and, importantly, to determine whether first-line standard therapies prescribed are effective against high-risk and invasive mammary tumours, given diverse tumour response and intrinsic tumour heterogeneity [7, 16–21].

Current prognostic biomarkers used in breast cancer
Clinicopathological parameters such as patient age, TMN (tumour size, lymph node status, metastasis) staging, tumour grade and histology, and molecular subtype of breast tumours have become commonplace in justifying medical decision making and prescribing treatment modalities. Therapies for invasive and high-risk breast cancer (luminal, basal-like, HER2-positive and triple-negative breast cancer) are routinely selected based on tumour ER, PR and HER2 status, in addition to aforementioned clinicopathological parameters [22–25]. The American Society of Clinical Oncology (ASCO) updated their clinical practice guidelines this year and according to the panel, only the current gold-standard biomarkers ER, PR, and HER2 should be used to guide treatment decisions, despite the fact they do not correlate with survival outcomes, nor predict tumour response to standard-of-care therapies [26].

ER-positive breast tumours account for the majority of cases worldwide [27]. Estrogen is the main stimulation for growth in the mammary tumours allowing this clinical biomarker to direct treatment planning and assess the benefit of mainstay anti-hormone therapies such as tamoxifen and aromatase inhibitors. The expression of PR is frequently associated with ER-positive tumours where less than 1% of mammary tumours are PR-positive and ER negative [27]. Furthermore, PR expression has shown strong prognostic value with high level PR expression in invasive breast tumours displaying better outcomes to anti-hormone treatment when compared to low level PR expression [28]. Current guidelines put forth by the ASCO recommend ER and PR testing of all new cases of primary and distant recurrent breast tumours [29].

HER2 status has been identified as a strong indicator of patient overall survival. Overexpression of the HER2 oncogene leads to higher chances of relapse with shorter survival times. The development of anti-HER2 therapies, such as trastuzumab, in treating HER2 have been shown to have major benefit and therefore HER2 status is tested in the majority of breast cancer diagnoses [30]. Other emerging biomarkers such as Ki67 have a possible prognostic role in invasive breast cancers [31]. While the exact function is unknown, Ki67 is expressed in cycling tumour cells and it has strong correlation with tumour grade [31, 32]. Approaches to combining Ki67 with established biomarkers ER, PR, and HER2 are now being used to further distinguish breast cancers into clinical subtypes. However, the ASCO did not recommend using Ki67 to guide adjuvant therapies [29].

The ASCO has recognized the clinical utility of six multigene expression assays for specific subgroups of patients, including OncotypeDX (Genomic Health), EndoPredict (Sividon Diagnostics), PAM50 (Prosigna Breast Cancer Prognostic Gene Signature Assay), Breast Cancer Index, urokinase plasminogen activator, and plasminogen activator inhibitor type 1 [33–37]. Most of these assays are recommended for patients with early-stage breast cancers only; none of these multi-gene assays were encouraged for patients with HER2-positive or triple-negative cancers, in addition to late-stage or metastatic cancers [34, 35]. The development of these promising gene signature-based molecular assessment tools for breast cancer is encouraging for the personalization of breast cancer therapies; however, there still remains an unmet medical need for patients with locally advanced and metastatic breast cancers.

The potential role of RAS pathway proteins in the prognosis and treatment of breast cancer
The oncogenic K-RAS carries the most common gain-of-function mutations in human cancer (30% of all human cancers) and its oncogenic role has been well-established in many types of human cancers [38, 39]. Thus, focusing on this tumour-promoting RAS signalling pathway is a logical choice of investigation for prognostic biomarker discovery and novel anticancer therapy development against oncogenic K-RAS-driven human cancer [40, 41]. The RAS pathway has been an intensely studied area of cancer research due to its activation of downstream effectors resulting in tumour proliferation, survival and motility (Fig. 1A). The loss of its most essential downstream signalling gatekeeper, SIAH (seven in absentia homologue), impeded oncogenic K-RAS-driven human pancreatic cancer and lung cancer [40, 42, 43]. Although oncogenic K-RAS mutations are rarely detected in mammary tumours (observed in about 5% of patients), genomic studies have indicated that the EGFR/HER2/K-RAS ‘pathway’ is activated in a large proportion of aggressive and malignant breast cancers [44, 45]. EGFR/HER2/K-RAS activation has been correlated with shortened survival, resistance to therapy, and tumour relapse despite aggressive treatments in breast cancer [44–47]. The mechanism and function of persistent RAS pathway activation remains elusive in breast cancer. This lack of mechanistic understanding, along with the low mutation rate of oncogenic RAS/RAF/MARK detected in mammary tumours, may contribute to the tumour-driving RAS pathway activation being understudied in the area of breast cancer research. It should also be noted that activation of RAS pathway in mammary tissues of animal models is adequate to induce oncogenic transformation and malignancy [48, 49]. Therefore, although there may not be any detectable RAS mutations in high-grade, locally advanced and metastatic breast tumours, alternative RAS pathway activation mechanisms of the RAS pathway downstream effectors may be present, promoting mammary tumorigenesis and metastasis.

Since RAS pathway activation is a major and essential signalling hub to promote human malignancies, we investigated whether EGFR/HER2/RAS pathway biomarker expression can be added to evaluate therapy efficacy and predict patient survival in breast cancer (Fig. 1A). It has been established that expression of SIAH, the most downstream signalling module and the most evolutionarily conserved component of the RAS signalling pathway, reflects RAS pathway activation, cell proliferation, and tumour growth [40, 42, 43]. In a retrospective study, 364 matched pairs of breast tumour specimens from 182 patients who received neoadjuvant systemic therapy (NST) were analysed to determine whether SIAH and/or EGFR outperform ER, PR, HER2, and Ki67 as a prognostic biomarker in locally advanced and metastatic breast cancer. The prognostic power of SIAH and EGFR, alone or in combination, is comparable to the clinical gold standards of clinical predictors (lymph node positivity, mammary tumour size, grade, stage and molecular subtypes in combination), and imaging-guided technology [50]. The activation/inactivation of the tumour-promoting RAS pathway biomarkers, SIAH and EGFR, is associated with tumour progression/regression in mammary tumours post-neoadjuvant systemic therapies (NST). We found that a marked reduction in SIAH/EGFR expression post-NST would indicate effective therapy and increased survival, while persistent high SIAH/EGFR expression post-NST would indicate ineffective therapy and decreased survival [50]. These results suggest that NST-induced reduction of SIAH and EGFR expression may be used as two useful surrogate prognostic biomarkers to quantify therapeutic efficacy, determine tumour responses, detect emerging resistant clones, and predict patient survival in high-grade and aggressive molecular subtype of breast cancer in the neoadjuvant setting in the future (Fig. 1B).

Conclusion
Early stage breast cancer is highly responsive to commonly prescribed standard of care therapies with excellent long-term survival. Locally advanced and metastatic breast cancer has a much worse prognosis despite aggressive chemo- and radiation therapies and locoregional surgical interventions. This disparity in prognosis underlines the acute need to tailor therapy and stratify patients in order to improve patient survival. The discovery of incorporating tumour heterogeneity-independent, therapy-responsive and tumour-driven RAS pathway biomarkers is prognostic in breast cancer, have a clear clinical impact to benefit breast cancer patients with locally advanced and metastatic diseases.

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The authors
Lauren L. Siewertsz van Reesema†*¹ BS; Michael P. Lee†¹ MS; Vasilena Zheleva² MD; Janet S. Winston³ MD; Caroline F. O’Connor4 MD; Roger R. Perry² MD, FACS; Richard A. Hoefer^5,6,7 DO, FACS; Amy H. Tang*^4,8 PhD
¹Eastern Virginia Medical School, Norfolk, VA 23507, USA
²Department of Surgery, Eastern Virginia Medical School, Norfolk, VA 23507, USA
³Sentara Pathology and Pathology Sciences Medical Group, Sentara Norfolk General Hospital (SNGH), Norfolk, VA 23507, USA
⁴Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
⁵Sentara Cancer Network, ⁶Dorothy G. Hoefer Comprehensive Breast Center, ⁷Sentara CarePlex Hospital, Newport News, Virginia 23606, USA
⁸Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA 23507, USA

†Authors contributed equally to this publication.
*Corresponding author
E-mail: Tangah@evms.edu

Recent years have witnessed the growing use of mass spectrometry (MS) in the clinical laboratory. MS provides massive improvements in the sensitivity and specificity of clinical tests. It does this by using an ionized molecule’s mass/charge (m/z) ratio for identification.
MS has its roots in the screening and fingerprinting of molecules in drugs of abuse. Over the years, the technology has rapidly evolved. Today, it is routinely used to screen for diseases and to precisely identify causes of infections for targeted therapies. The analysis of proteins is also accelerating, with special potential demonstrated by biomarkers such as thyroglobulin.Alongside, limitations to immunoassays have also driven adoption of MS. For example, no immunoassays were approved for the immunosuppressant sirolimus and this compelled laboratories to turn to MS. Another advantage lay in improved assay quality, such as the measurement of testosterone in patients with low endogenous concentrations, such as women and children.

Enticing advantages
One of the most enticing set of advantages of mass spectrometry is that it provides clinically relevant information from relatively small sample volumes, and does this both rapidly and at a reduced cost. Gas chromatography (GC), liquid chromatography (LC) and ion mobility spectrometry (IMS) separation now allow targeting of ever-smaller analyte concentrations. LC-MS/MS (liquid chromatography-tandem mass spectrometry), on its part, offers scope to cut costs further, while continuing to improve accuracy. Other technology trends include integrating MS with low-flow chromatography, ultra-high pressure chromatography and online/multi-dimensional  chromatography.
Nevertheless, MS also adds a new layer of complexity. As a result, close and well-structured communication between laboratories and clinicians is a vital component for the effective use of MS.

A three-step process
Today, there are three principal steps for conducting an analysis by MS: sample preparation, separation by gas-chromatography (GC) or liquid-chromatography (LC), and mass spectrometric analysis.
In MS, a sample ‘matrix’ refers to everything present in a sample, excluding analytes of interest. Differences in behaviour between analytes and matrix components determines the choice of sample preparation. Although sample preparation requires more labour than immunoassays, in-house mass spectrometry-based assays are now considered cost-effective, even for smaller labs.

Sample preparation
The preparation of samples and their subsequent separation by chromatography both use mechanisms which first position molecules (the stationary phase) and then separate analytes from matrix components (the mobile phase).
Preparation firstly depends on the sample type selected for analysis (e.g. blood/serum or urine). Analytes from serum (including blood fractions) require the maximum care in preparation, owing to a relatively low ratio in the concentration of analytes to matrix components. On the other hand, urine analytes are often compatible with simple dilution protocols, due to the concentrating effect of kidneys in the production of urine.

Typical techniques in preparing samples include solid-phase extraction (SPE), immunoextraction and dilution. The choice depends principally on whether the analytes are acidic or alkaline, and if they are heavily protein-bound.

Solid-phase extraction
SPE is based on combining a solid stationary phase with a liquid mobile phase.
Analytes of interest (and matrix components) remain in the liquid phase or associate only temporarily with the solid stationary phase. The amount of time taken up by the latter is based on characteristics such as charge and polarity of the analytes versus matrix components. A binding-and-wash solvent (different from the elution solvent), provides a relatively crude separation of analytes from the (unwanted) components.

Immunoextraction
Immunoextraction (also known as immunoaffinity purification) is based on the use of antibodies in a solid phase. This separates antibody-bound analytes from free matrix components.

Dilution
When analytes are present in high concentrations, dilution provides a simple and effective methodology to reduce matrix components. Dilution (often called ‘dilute-and-shoot’) is a common method of sample preparation for comprehensive screening and for confirmatory testing for drugs in urine.

Separation
Gas chromatography
GC chromatography uses hydrogen or helium to push molecules into a column (known as the stationary phase). Modifying the column temperature then changes the affinity of molecules in the stationary phase, thereby separating analytes from matrix components (known as the mobile phase). Though largely relevant for volatile, heat-stable compounds, ‘derivatization’ via chemical modification can increase compatibility with GC.
GC mass spectrometry (GC-MS) remains the most common method for comprehensive drug screening in the clinical laboratory.

Liquid chromatography
LC chromatography is largely used for separation of samples before MS analysis. This is largely due to a wide range of LC-compatible analytes and a reduced need for derivatization. The mobile phase in LC uses a combination of organic solvents and water. Adjustments to the ratio between the organics and water redistributes components between the mobile and stationary phases.

Ionization techniques: APCI and electrospray
MS detects charged analytes in the gaseous phase alone. Ionization is required to convert liquid-phase analytes for analysis. The two most common methods in the clinical laboratory consist of atmospheric pressure chemical ionization (APCI) and electrospray ionization.
APCI produces ions by using heat to evaporate the solvent and plasma to ionize the sample. Physical interaction with gaseous analytes leads to formation of negative or positive ions.
Electrospray ionization, on its part, combines electricity, air and heat to produce successively smaller and concentrated droplets from the liquid which elutes off a chromatographic column. This leads to a dramatic increase in charge per unit volume. Ions on the droplet surface desorb from liquid to gas phase, and the latter is introduced into the mass spectrometer.

Sample transfer to mass spectrometer
There are several choices for introducing samples into a mass spectrometer. These range from direct infusion to multidimensional chromatographic separation. The latter enables the staggered delivery of analytes and matrix components. This permits more effective utilization of analyser time by limiting analysis to fractions containing analytes of interest.

Methods of analysis
MS analysis is largely based on quadrupole analysers, time-of-flight (TOF) analysers and tandem mass spectrometers, as well as combinations of the three.

Quadrupole analysers
Linear quadrupole analysers are currently the most common type of mass spectrometer in a clinical laboratory. Called quadrupoles due to the presence of four parallel rods in a square, one pair of (diagonally opposed) rods is positively charged, while the other is negatively charged. The charges are optimized and alternated based on the analyte of specific interest. Via sequential attraction and repulsion, an ion of interest can be programmed to maintain a stable flight path between the rods. The charge and frequency can moreover be rapidly altered to sequentially detect different analytes. Quadrupole analysers have high sensitivity and mass accuracy. On the other hand, they have a limited range in mass/charge (m/z) ratios – which, as noted previously, is a unique identifier for a particular ion.

Time-of-flight analysers
Time-of-flight (TOF) mass spectrometers are based on using an electric field which accelerates gas phase ions to a detector. The time taken for this travel is based on an ion’s m/z ratio, with low m/z ions travelling faster than higher ones.
TOF analysers have an essentially unlimited m/z range and high sensitivity and accuracy, but users face limits in their dynamic range.

Key challenges in clinical MS
Tandem mass spectrometry
Successful identification of an analyte by m/z alone does not always confer specificity. One good example is morphine and hydromorphone. Though the two are distinct, they have identical positive ions, with 286 m/z.
Tandem mass spectrometers (MS/MS) use multiple quadrupoles in series. One typical configuration is to use three quadrupoles. The first and third (denoted Q1 and Q3) use combinations of charge and frequencies as described above (see section on ‘Quadrupole Analysers’). The second quadrupole, denoted q2 (in smaller case), serves as a collision cell with an inert gas (e.g. nitrogen). On entry into q2, ions collide with the inert gas, and fragment into smaller product ions which then pass through Q3 and hit a detector.
In the case of morphine and hydromorphone, q2 entry produces stable product ions (m/z of 153 for the former and 157 for the latter). After this, setting the Q3 charge and frequency to first transmit the product ion for morphine and then change the charge/frequency settings to transmit the  hydromorphone product ion results in a way to measure and differentiate between the two compounds. This approach is also known as multiple reaction monitoring (MRM) and allows a mass spectrometer to scan faster – by targeting specific m/z set points rather than a broad range.

Ion suppression/enhancement and ion ratios
Ion suppression and enhancement are two of the most common problems facing MS. These occur when a substance in a sample interferes with the ionization process of the analytes. These can range from matrix molecules to co-eluting compounds. For example, components in the sample with lower volatility can reduce the efficiency of solvent evaporation, resulting in reduced ion formation.
There are several options to reduce or eliminate such interference, including mobile phase additives to aid ionization.
Another approach is to use ion ratios. When analytes of interest are present alongside structurally similar compounds in complex matrices, interference risks rise due to co-eluting molecules with identical mass. However, ion ratios seek to monitor multiple m/z transitions for each analyte and determine ratios of chromatographic peak area for more abundant fragments to less abundant ones. The use of ion ratios further enhances the specificity of MS.

The future
Endocrinology
Although industry has sought to use antibody-mediated detection to overcome the inherent limitations of immunoassays in identifying proteins and small molecules, these have yet to be meaningfully eradicated.
Rising interest in (regular and more-frequent) testing for vitamin D has also driven implementation of LC-MS/MS (liquid chromatography-tandem mass spectrometry), which separates vitamin D2 from vitamin D3 and provide information on its epimeric form. This is not possible with existing immunoassays.
Meanwhile, although steroid hormone assays for diagnostic and forensic testing continue to grow, a lack of specificity and accuracy at low concentrations has hampered the diagnosis of endocrine disorders. This has led several medical groups to recommend mass spectrometry as the preferred method of analysis, in spite of the high degree of technical competence, skill and experience required to achieve meaningful results.

Metabolomics
Measurements of the genome and proteome need to be accompanied by quantified data on the metabolome to comprehend differences between disease and healthy status, and provide meaningful diagnosis and monitoring of disease. One of the fastest growing areas for MS in metabolomics is the screening of newborns.

Protein analysis
The success of MS in precise measurement of small molecules has driven interest in using it for peptide and protein analysis for diagnostic testing. In spite of some challenges, quantitative proteomics (covering factors such as isotope dilution and m/z transitions) is an especially exciting application for mass spectrometry.

Indirect immunofluorescence (IIF) is an indispensable method for autoantibody diagnostics, providing high sensitivity and specificity together with a broad antigenic spectrum. However, the microscopic evaluation of the fluorescence patterns is both time-consuming and challenging for laboratory staff, and is, moreover, based on subjective interpretation. Laboratories are increasingly turning to automated systems to facilitate and standardize the IIF readout and interpretation. In recent years various automation systems have been developed, which provide automated digital acquisition of IIF images, discrimination of positive and negative samples, as well as pattern classification for key applications. This article focuses on the EUROPattern system, which provides computer-aided immunofluorescence microscopy for anti-nuclear antibodies (ANA), anti-neutrophil granulocyte cytoplasm antibodies (ANCA), antibodies against double-stranded DNA (anti-dsDNA) on Crithidia luciliae, monospecific antigen microdots (EUROPLUS) and transfected cell-based assays e.g. for anti-neuronal antibodies. The accuracy of automated evaluation compared to visual assessment has been investigated in various published studies.

by Dr Jacqueline Gosink

ANA
ANA represent a key diagnostic criterion for many autoimmune diseases, including systemic lupus erythematosus (SLE), mixed connective tissue disease, Sjögren’s syndrome, systemic sclerosis, polymyositis, dermatomyositis and primary biliary cirrhosis. The gold standard for ANA determination is IIF on human epithelial (HEp-2) cells. This substrate provides the complete antigen spectrum and allows investigation of over 100 different autoantibodies. Observation of the fluorescence pattern enables classification of the antibody or antibodies present in the patient sample. Positive results are confirmed by monospecific tests such as ELISA, immunoblot or IIF microdot assays.

The automated evaluation of HEp-2 cells includes reliable discrimination of positive and negative ANA results, as well as classification of all ANA patterns [1] (Figure 1), encompassing homogeneous, speckled, nuclear dots, nucleolar, centromeres, nuclear envelope and cytoplasmic. The ANA patterns identified by EUROPattern correspond to the competent level reporting defined by the International Consensus on ANA Patterns (ICAP; www.anapatterns.org). Mixed patterns, which occur when more than one antibody is present, are also recognized and reported as such. The pattern is assigned by analysing its features and comparing it to a reference database of over 5000 images, corresponding to 115,000 cells. Unspecific signals originating from outside of the cells are identified by means of a DNA counterstain and subsequently rejected. The evaluation also includes titre designations with confidence values for the detected antibodies. Results from the HEp-2 screening can be monospecifically confirmed using microdot substrates of purified antigens, which are incubated and evaluated in parallel.

To assess the diagnostic accuracy, the automated evaluation was compared to conventional visual interpretation by experts in the field using 351 patient sera [2]. The concordance for positive/negative discrimination was 99%, with an analytical sensitivity of 100% and a specificity of 98%. In 60% of samples, the pattern, including variable mixed patterns, was recognized completely by the software. In 94% of samples, the main pattern was correctly designated. A further study showed 79% correct pattern assignment.

Anti-dsDNA antibodies
Anti-dsDNA antibodies are a hallmark of SLE and represent an important criterion for diagnosis. Their prevalence in SLE ranges from 30% to 98% in different studies, depending among other things on the test method used. Like the gold standard Farr assay, IIF using Crithidia luciliae as the substrate (CLIFT) is considered to have a very high disease specificity. The method takes advantage of the kinetoplast of C. luciliae, which is rich in DNA but contains hardly any other antigens, thus enabling highly selective detection of anti-dsDNA antibodies. However, manual reading of the fluorescence signals is subjective and leads to high intra- and inter-laboratory variation, making standardized automated evaluation a desirable goal.
Automated interpretation of CLIFT has recently been incorporated into the EUROPattern system [3] (Figure 2). The software is able to recognize the organelles of the protozoan and evaluates the specific kinetoplast fluorescence rather than just dark-light classification, increasing the reliability of the evaluation. Results are classified as positive or negative, and include a titre designation based on the fluorescence intensity.

In a clinical study, automated and visual evaluation of C. luciliae IIF was compared using 569 consecutive sera submitted for routine anti-dsDNA screening and 100 sera from healthy blood donors. The automated system recognized all 73 of the anti-dsDNA positive samples identified by the visual evaluation. Moreover, 93% of the titre designations were concordant. The overall sensitivity of the system amounted to 100% with a high specificity of 97%. Compared to visual microscopy the overall accuracy was 97%.

ANCA
ANCA are important serological markers for diagnosis and differentiation of autoimmune vasculitides, especially granulomatosis with polyangiitis (GPA, formally known as Wegener’s granulomatosis), which is characterized by autoantibodies against proteinase 3 (PR3), and microscopic polyangiitis, which is typified by autoantibodies against myeloperoxidase (MPO). In addition, ANCA can be found in chronic inflammatory bowel diseases. ANCA are detected by IIF with monospecific confirmation using ELISA, immunoblot or IIF microdot assays.
The IIF substrates ethanol-fixed and formalin-fixed granulocytes are used to identify the typical ANCA staining patterns of anti-PR3 (cytoplasmic, cANCA) and anti-MPO (perinuclear, pANCA) antibodies. An additional substrate consisting of HEp-2 cells coated with granulocytes allows immediate differentiation between ANCA and ANA, while purified antigen microdots of PR3, MPO or glomerular basement membrane (GBM) antigen provide simultaneous monospecific antibody characterization. The different substrates are incubated and automatically evaluated in parallel as BIOCHIP mosaics, thus providing ANCA screening and confirmation in one step.

Evaluation software such as EUROPattern provides automated positive/negative discrimination of samples, as well as recognition of pANCA and cANCA patterns [1] (Figure 3). Further pattern constellations such as DNA-ANCA (atypical pANCA, xANCA), which can arise from antibodies against lactoferrin or other antigens, are also taken into account by the software. The automated system proposes a result based on the recognized cellular patterns and the results on the antigen microdots. An estimated titre with a confidence value is given.

Anti-neuronal antibodies
Neuronal cell-surface autoantibodies occur in autoimmune encephalitis and their detection can secure an early diagnosis, enabling immediate treatment which is critical for patient outcome. In recent years a considerable number of novel target antigens has been discovered, for example, glutamate receptors of type NMDA and AMPA, GABAB receptors, voltage-gated potassium channel-associated proteins LGI1 and CASPR2, DPPX and IgLON5.

Diagnostic tests for the new parameters are based on recombinant-cell (RC) IIF, in which transfected cells expressing the relevant antigen are used for monospecific antibody detection. This test method enables authentic presentation of the fragile membrane-associated surface antigens. Since many of the autoantibody markers are rare and do not always overlap, a multiparametric screening using BIOCHIP mosaics made up of different substrates is recommended. Results for RC-IIF assays can be evaluated automatically using a newly developed function of EUROPattern. The system automatically takes digital images of the substrates and provides a positive/negative classification.
The quality of the acquired images was assessed by comparing on-screen appraisal with visual microscopy using 753 incubations of numerous serum samples sent to a clinical immunology laboratory [4]. Ambiguous fluorescence signals detected at the microscope were excluded to avoid inter-reader deviations. The two evaluation strategies revealed a concordance of 100% with respect to positive/negative discrimination, confirming the high quality of the images.

Arbovirus antibodies
Immunofluorescence microscopy is also useful for infectious disease diagnostics. For example, infections with Zika virus, dengue virus and chikungunya virus are difficult to tell apart clinically as they manifest with similar symptoms and are endemic in much the same regions. Serological tests are an important diagnostic method, especially beyond the short viremic phase when direct detection is no longer effective. Viral antibodies can be detected by IIF using virus-infected cells. However, cross reactions between flavivirus antibodies can occur.

A BIOCHIP mosaic comprising substrates for Zika, dengue and chikungunya viruses enables parallel antibody determination, aiding clarification of cross reactivities and supporting differential diagnosis. The substrates can be evaluated semi-automatically using the digital image acquisition function of EUROPattern. In particular, inspection of the images side-by-side on the computer screen considerably facilitates the interpretation.

Fully automated immunofluorescence microscopy
Computer-aided fluorescence microscopy can be further standardized and facilitated through use of complementary hardware. The EUROPattern microscope (Figure 4) has been tailored to the requirements of immunofluorescence. Next to the high-precision optical system, it has a controlled LED, which maintains a constant light flux, ensuring highly reproducible results. The cLED has an extremely long life span without maintenance (over 50,000 hours) and low power consumption, ensuring cost-effectiveness for laboratories. The microscope is equipped with a slide magazine which can process up to 500 analyses in succession within 2.5 hours (18 seconds per field), correctly identifying the slides by means of matrix codes.

Results from the automated IIF evaluation can be viewed and validated directly at the computer screen, enabling a diagnosis to be established quickly and efficiently. The high-resolution images are sharply focused with the aid of a counterstain. The counterstain also serves to verify correct performance of the incubation. Negative results can be verified in batches, while positive samples can be individually checked and confirmed by the medical technologist. Results from different serum dilutions and substrates are consolidated into one report per patient, and new findings are compared with previous records. Final results can be signed electronically and forwarded at a click.

Perspectives
The need for standardization and automation in IIF is tremendous in all fields of autoimmune diagnostics. In particular, manual evaluation of results is time consuming and subjective. Automation platforms with harmonized software and hardware components have in recent years contributed enormously to the standardization and simplification of the evaluation process, especially for ANA, ANCA and CLIFT. Advanced software provides positive/negative classification, pattern recognition and titre designation at a quality equivalent to visual microscopy. The recording of tissue substrates, such as liver, kidney, stomach, esophagus, small intestine, heart and neuronal tissue, is also feasible. Future development will focus on the recognition of organ- and non-organ-specific autoantibodies on tissues, for example antibodies against mitochondria, epithelial membrane, epidermal basement membrane, desmosomes, heart muscle and neuronal antigens. The continued development of automated evaluation systems is anticipated to lead to even greater standardization of IIF and further reductions in workflow for diagnostic laboratories.

References
1. Krause et al. Lupus 2015: 24: 516-29
2. Voigt et al. Clin. Devel. Immunol. 2012: vol 2012, article ID 651058
3. Gerlach et al. J. Immunol. Res. 2015: vol 2015, article ID 742402
4. Fraune et al. Autoimmunity Reviews 15 (2016) 937-942

The author
Jacqueline Gosink, PhD
EUROIMMUN AG, Seekamp 31, 23560 Luebeck, Germany
E-mail: j.gosink@euroimmun.de

Urinalysis may provide evidence of significant renal disease in asymptomatic patients. The microscopic urinalysis is vital to making diagnoses in many asymptomatic cases, including urinary tract infection, urinary tract tumors, occult glomerulonephritis, and interstitial nephritis.

Presence or absence of different particles in urine sediment is crucial for clinical decision making. Urine sediment cells or particles provide important information for the diagnosis of renal or urinary diseases [1]. The patented UriSed Technology was developed to reduce the shortcomings of manual microscopy through automation. [2]. The UriSed analysers provide a reliable and reproducible solution since 2007 [3]. The new generation instrument based on the improved UriSed Technology, UriSed 3 was introduced in the market in 2015. UriSed 3 is an automated urine microscopy analyser with a revolutionary particle visualization utilizing both bright-field and phase-contrast microscopy. In the present study, we evaluated the analytical performance of UriSed 3 Automated Urine Microscopy Analyser (Manufactured by 77 Elektronika Kft., Budapest) and compared the results to those from manual microscopy using standardized KOVA counting chambers.

UriSed 3 provides quantitative Red Blood Cell (RBC) and White Blood Cell (WBC) results, and semi-quantitative results for all other particle types: WBC Clumps (WBCc), Squamous Epithelial Cells (EPI), Non-squamous Epithelial Cells (renal tubular and urothelium cells) (NEC), Crystals (CRY): Calcium oxalate dihydrate (CaOxd), Calcium oxalate monohydrate (CaOxm), Uric acid (URI), and Triple-phosphate crystals (TRI), Hyaline casts (HYA), Pathological casts (PAT), Bacteria (cocci-like and rod-like) (BACc, BACr), Yeasts (YEA), Spermatozoa (SPRM) and Mucus (MUC) [4].

Phase-contrast microscopy by UriSed 3
Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. In particular, for urinary sediment examination, phase-contrast supplies an optimal identification of particles with a low refractive index (e.g., hyaline casts and RBC devoid of their hemoglobin content, the so-called “ghost RBC”) and of cellular morphological details) [9, 10]. Therefore the use of phase-contrast microscopy is encouraged also by international guidelines on urinalysis [5, 6].

The measurement technique of the UriSed 3 instrument is a combination of a bright-field microscope and a phase-contrast microscope in one optical system. Preparation of the UriSed 3 analyser for measurement takes only a few minutes. It needs distilled water for washing its pipette, and patented disposable plastic cuvettes for sample investigation. The instrument throughput is up to 120 samples per hour. The whole measurement process is completely automatic: 200 µl of urine sample is dispensed into the cuvette, then spinning the cuvette for a few seconds gently deposits formed elements into a monolayer at the bottom of the cuvette. The built-in digital camera takes and saves both a bright-field and a phase-contrast microscopic image from the same view-field at 15 different positions of the sediment layer. Information from both whole view-field images are evaluated by a neural network based image processing software.

Material and methods
Analysis of 311 samples was performed to evaluate UriSed 3 analytical performance compared to the manual microscopy urine examination method. Both measurements were carried out with the same anonymous urine samples. Fresh, native urine samples were used, that were typically held for no more than 4 hours before being analysed, as recommended in the relevant guidelines [5,6] to prevent change in the morphology of the particles. Samples were mixed until homogeneous and then split and run on each measuring procedure as close to the same time as possible. The standardized microscopic urinalysis of native samples (Level 3) was followed by using a KOVA counting chamber. The particle concentration for all particle types was evenly distributed in the evaluated urine samples. Carry-over, precision, diagnostic tests such as sensitivity, specificity, diagnostic accuracy, concordance and one category concordance were investigated according to well-established protocols [7].

Results
No carry-over was detected in any of the samples. UriSed 3 has better precision than microscopy at all of the tested RBC and WBC concentrations. The majority of all coefficients of variation obtained for within series imprecision (CV) using UriSed 3 was 7-16% versus 5,5-67% in case of manual microscopy [8]. Good correlation can be found between UriSed 3 and manual counting chamber for formed elements. The Pearson correlation of quantitative parameters are 0.91 (RBC), 0.93 (WBC). The clinical evaluation of UriSed 3 was based on McNemar test and concordance study. The results are shown in the table above.

Conclusion
UriSed 3 instruments utilize phase-contrast and bright-field microscopy to combine original and innovative technologies whose aim is the progressive improvement of automated urinary sediment examination and the progressive approach to the gold standard manual microscopy method. The automated measurement process of UriSed 3 is reproducible and operator-independent. Those sediment particles that are mostly transparent become visible with phase-contrast microscopy by UriSed 3, which is a spectacular advantage and leads to specific improvement in recognition at several particle types.

References
1. Fogazzi GB, The Urinary Sediment an Integrated View Third Edition. Milano: Elsevier, 2010.
2. Barta Z, Kránicz T, Bayer G. UriSed Technology – A Standardised Automatic Method of Urine Sediment Analysis. European Infectious Disease 2011;5:139–42.
3. Zaman Z, Fogazzi GB, Garigali G, Croci MD, Bayer G, Kránicz T. Urine sediment analysis: analytical and diagnostic performances of sediMAX – a new automated microscopy image-based urine sediment analyser. Clin Chim Acta 2010; 411: 147-154.
4. Fogazzi GB, Garigali G. The Urinary Sediment by UriSed Technology. A New Approach to Urinary Sediment Examination. Milano: Elsevier, 2013.
5. Kouri T, Fogazzi G, Hallander H, Hofmann W, Guder WG, editors. European Urinalysis Guidelines. Scand J Clin Lab Invest 2000; 60 (Suppl 231): 1-96.
6. Clinical and Laboratory Standard Institute (ex NCCLS). Document GP16-A3 – Urinalysis; Approved guideline, 3rd ed. Wayne, PA: CLSI, 2009.
7. T. Kouri, A. Gyory, R.M. Rowan. ISLH Recommended Reference Procedure for the enumeration of Particles in Urine. Laboratory Hematology 9:58-63, 2003.
8. Haber MH, Galagan K, Blomberg D, Glassy EF, Ward PCJ, editors. Color Atlas of Urinary Sediment; An Illustrated Field Guide Based on Proficiency Testing. Chicago: CAP Press, 2010.
9. Brody L, Webster MC, Kark RM. Identification of elements of urinary sediment with phase-contrast microscopy. JAMA 1968; 206: 1777-1781.
10. Spencer E. and Pedersen Ib. Hand Atlas of the Urinary Sediment. Bright-field, Phase-Contrast, and Polarized Light. Copenhagen: Munksgaard, 1971.

More information on UriSed 3 is available from the manufacturer:
77 Elektronika Kft., Budapest, HUNGARY
Email: sales@e77.hu, web: en.e77.hu

The author
Erzsébet Nagy MD,
Honorary Associate Professor
Head Phisician of Central Laboratory; Hospitaller Brothers of St. John of God Hospital, Budapest