Point-of-care testing (POCT) enables quick test results with minimal manual interference nearer to the site of patient care, which leads to better health outcomes via rapid diagnosis, quick clinical decisions and the early start of treatment. The emerging technologies would further improve POCT by low-cost analysis with increased performance characteristics.

by Dr Sandeep Kumar Vashist and Prof. John H.T. Luong

Existing point-of-care testing (POCT) technologies
A variety of POCT technologies are being used such as POCT analysers, biosensor devices, lab-on-chips (LOC), test strips, and lateral flow assay (LFA) cartridges. Such cost-effective technologies offer rapid analysis in just a few minutes using minimal sample volumes. As well as at a patient’s bedside, POCT have been employed in the operating theatre, emergency department and critical care/maternity unit. Other deployments include nursing homes, physician’s office, prison, emergency vehicles, etc. Local pharmacies have adopted the POCT technology to provide a one-stop service for glucose, cholesterol, pregnancy, etc.

The POCT analysers are standard bench-top devices that can determine a broad range of analytes, based on spectrophotometry, reflectometry, immunoassay, turbidimetry, potentiometry/amperometry, oximetry and hematological particle counting. The target analytes are small metabolites, enzymes, drugs-of-abuse, inflammation biomarkers, heart and kidney injury biomarkers, infectious agents, humoral and cellular coagulation markers, hematological parameters, etc.

The biosensor-based devices, notably the blood glucose meters, are the most common POCT technology, which have been widely used for the detection of glucose. Different LOC platforms are being used in various POCT technologies. They are fully automated platforms that integrate all microfluidics-based bioanalytical steps such as sample treatment, separation, biomolecular detection, washing, signal detection, and data processing, storage, and transmission. The signal detection in most LOC platforms employs optical readout. The technologies and materials used for the production of LOC platforms have been fully characterized and standardized.

Test strips are another prominent POCT technology for detecting different analytes in a patient’s blood or urine sample. They are easy to use and easy to read, leading  to immediate on-the-spot analysis. The test strip comprises a solid support onto which porous matrices with dried assay reagents are integrated. The reaction starts as the biorecognition element present on the test strip detects the analyte, which leads to a visual change in colour on the test strip. The signal is read by inserting the test strip into a reader device.

LFA, one of the most widely used POCT technologies, is based on an immunochromatography format where the assay reagents are stored in dried form on various porous materials. Once the sample (urine or diluted blood) is dispensed at the designated area of the LFA cartridge, the sample flows in the lateral direction by capillary forces. It first interacts with the capture antibodies spotted at the reaction area leading to the formation of the immune complex, which is followed by binding to the detection antibodies immobilized at another area of the cartridge, thereby resulting in the formation of the sandwich immune complex. This leads to a visible colour change in the test and control lines, which facilitates rapid qualitative or semi-quantitative analysis. The POC pregnancy testing is done exclusively by LFA.

The multiplex analysis using DNA and protein microarrays is also being intensively investigated although the technology has not yet been commercialized for clinical POCT. It is envisaged that this upcoming technology would be fully automated, which would involve advanced microfluidics and the signal readout by electrochemical, chemiluminescence, fluorescence or evanescent wave techniques.

Emerging POCT technologies
Various POCT technologies have emerged during the last decade, which have tremendous potential for next-generation healthcare monitoring and management [1]. In 2011, the estimated total POCT market was US$15 billion and projected to reach US$18 billion by 2016. Of the total POCT market in 2011, 55% of it was in the US market, followed by 30% in Europe and 12% in Asia [2].

Cellphone (CP)-based devices
The most prospective emerging technology is CP-based devices. CPs have become ubiquitous with more than 7 billion global users that account for more than 95% of the world’s population. Moreover, about 70% of the CP users reside in the developing countries, where there is an imminent need for mobile healthcare (mH). The current generation of CPs are cost-effective and equipped with all the desired advanced features that facilitate personalized mH monitoring and management. The spatiotemporal tagging of the data by CP enables the real-time active response to epidemics and emergency situations. Various FDA approved and CE certified CP-based personalized healthcare devices have already been commercialized for the monitoring of basic physiological parameters such as blood glucose, blood pressure, pulse rate, blood oxygen saturation, body weight, body analysis parameters, electrocardiogram, physical activity, sleep and cardiac parameters (Fig. 1) [3]. Most of such commercial CP-based devices are developed by iHealth Labs, France. Similarly, CP-based technologies have been prepared for many POCT applications [4]. Cellmic, USA has developed a compact CP-based rapid-diagnostic-test reader for the readout of colorimetric and fluorometric LFA (Fig. 2). Of notice is the conversion of the CP into a compact and lightweight computational microscope for bright field, fluorescence, darkfield, transmission and polarized microscopy modes. Moreover, a CP-based flow cytometer based on optofluidic fluorescent imaging enabled the screening of pathogens in whole blood or water samples [5]. Another similar endeavour is the development of smartphone-based spectrophotometers for the detection of absorbance, fluorescent or chemiluminescent signals [6]. Various CP-based colorimetric readers [7], electrochemical sensing platform [8], angle-resolved surface plasmon resonance (SPR) system [9], and multiplex assays have also been developed [10].

Paper-based diagnostics
Considering simplicity and cost-effectiveness, paper-based diagnostics (PBD) are available in various formats: LFA, dipstick and microfluidic paper-based analytical devices (µPADs) [11, 12]. LFA are widely used in home pregnancy test strips to detect human chorionic gonadotropin in urine. It employs the dispensing of the sample onto the sample pad of the LFA test strip (fabricated from a nitrocellulose membrane). The sample flows laterally over a conjugate pad due to the capillary action provided by the absorbent pad, which leads to the binding of the analyte to conjugate particles (gold nanoparticles and upconversion nanoparticles). The signal detection can be visual, colorimetric, electrochemical, photoelectrochemical, chemiluminescent and electrochemiluminescent. The colorimetric PBD provide qualitative analysis by comparing the colour against a predetermined score chart. But the colour intensity can also be quantified using cameras, scanners, commercial test strip readers and hand-held colorimeters. A CP-based rapid-diagnostic-test reader is the most recent development that enables precise determination of colour intensity [13].

Paper can be patterned to fabricate two-dimensional (2D) or three-dimensional (3D) µPADs. The 3D µPADs, formed by stacking layers of the 2D paper, are ideal for multiplexing. The microfluidic paper-based electrochemical devices (µPEDs) can be fabricated by printing electrodes on paper.

The sensitivity of PBDs can be increased by employing enzymes and nanomaterials based signal enhancement strategies, which increases the costs and assay duration, and decreases the shelf-life. However, PBDs suffer from poor reproducibility, non-uniformity and variable accuracy on µPADs due to the passive capillary transport in paper substrates.

Lab-on-a-chip platforms
Various LOC platforms, such as the most widely used blood glucose testing strips, have been made for POCT. These platforms enable fully automated analysis by integrating all process steps in the operational procedure. The Piccolo Xpress™ whole blood chemistry analyser, developed by Abaxis Inc, is a prospective LOC-based POCT device that performs 14 tests on a single reagent LabDisk (8 cm diameter, barcoded).

Rapid assay formats
A prospective assay format is Optimiser™ ELISA [14] by Siloam Biosciences Inc,  which employs a novel microfluidic microtiter plate. It detects an analyte in just a few minutes using minimal reagent volumes and least number of steps. Other prospective formats are the wash-free AlphaLISA^® by Perkin Elmer, wash-free electrochemiluminescent ELISA by Meso Scale Diagnostics LLC, rapid one-step kinetics-based formats [15], CP-based easy immunoassay platforms [16], and COBAS^® Lab-in-a-Tube (LIAT) system by Roche Diagnostics.

Prolonged reagent storage strategies
The most prospective prolonged reagent storage strategies are the use of polysaccharides and saccharides (such as pullulan and trehalose), sugar alcohols, stabilizers, freeze drying, lyophilization, and reagent pouches. Use of POCT in remote areas, particularly in developing countries might be problematic due to inconsistent electrical power, lighting, and refrigeration.

Conclusions
The next decade will witness the revolutionary breakthroughs in POCT that will drastically cut down the costs and lead to considerably improved analysis with increased bioanalytical performance and capabilities. Multi-channel, high-throughput instruments are expected to expand the new concept of POCT, and there is an obvious need for the combination of POCT technology and communication technology. Non-invasive blood glucose monitoring remains the most important market for POCT, followed by coagulation, blood gas, chemistry, hematology, urinalysis, and cardiac. Also, molecular POCT technology has matured and began to move toward commercialization.

Future trends for POCT will rise due to new emerging technologies to make them well suited for low-resource or remote areas. POCT vendors increasingly offer more available types of tests with improved accuracy and minimal turnaround times. Thus, POCT will be more widely accepted as an addition to the current method of managing patients. Considering its limited test menus, clinicians still have to wait for other sophisticated laboratory tests before the action can be taken for treatment or further testing [17]. POCT evolves continuously and becomes an ‘extension’ of laboratory services, not a replacement of routine core laboratory testing.

References
1. Vashist SK, Luppa PB, Yeo LY, Ozcan A, Luong JHT. Emerging technologies for next-generation point-of-care testing. Trends Biotechnol. 2015; 33(11): 692–705.
2. Scientia Advisors. The point-of-care diagnostics market. 2013, Cambridge, MA, USA. http://www.businesswire.com/news/home/20100915005199/en/Scientia-Advisors-Projects-Sustainable-Growth-Point-of-Care
3. Vashist S, Schneider E, Luong JHT. Commercial smartphone-based devices and smart applications for personalized healthcare monitoring and management. Diagnostics 2014; 4(3): 104–128.
4. Vashist SK, Mudanyali O, Schneider EM, Zengerle R, Ozcan A. Cellphone-based devices for bioanalytical sciences. Anal Bioanal Chem. 2014; 406(14): 3263–3277.
5. Zhu H, Mavandadi S, Coskun AF, Yaglidere O, Ozcan A. Optofluidic fluorescent imaging cytometry on a cell phone. Anal Chem. 2011; 83(17): 6641–6647.
6. Yu H, Tan Y, Cunningham BT. Smartphone fluorescence spectroscopy. Anal Chem. 2014; 86(17): 8805–8813.
7. Vashist SK, van Oordt T, Schneider EM, Zengerle R, von Stetten F, Luong JHT. A smartphone-based colorimetric reader for bioanalytical applications using the screen-based bottom illumination provided by gadgets. Biosens Bioelectron. 2015; 67: 248–255.
8. Lillehoj PB, Huang MC, Truong N, Ho CM. Rapid electrochemical detection on a mobile phone. Lab Chip. 2013; 13(15): 2950–2955.
9. Preechaburana P, Gonzalez MC, Suska A, Filippini D. Surface plasmon resonance chemical sensing on cell phones. Angew Chem Int Ed Engl. 2012; 51(46): 11585–11588.
10. Laksanasopin T, Guo TW, Nayak S, Sridhara AA, Xie S, Olowookere OO, Cadinu P, Meng F, Chee NH, et al. A smartphone dongle for diagnosis of infectious diseases at the point of care. Sci Transl Med. 2015; 7(273): 273re1.
11. Mao X, Huang TJ. Microfluidic diagnostics for the developing world. Lab Chip 2012; 12(8): 1412–1416.
12. Li X, Ballerini DR, Shen W. A perspective on paper-based microfluidics: current status and future trends. Biomicrofluidics 2012; 6(1): 11301–11301-13.
13. Mudanyali O, Dimitrov S, Sikora U, Padmanabhan S, Navruz I, Ozcan A. Integrated rapid-diagnostic-test reader platform on a cellphone. Lab Chip 2012; 12(15): 2678–2686.
14. Kai J, Puntambekar A, Santiago N, Lee SH, Sehy DW, Moore V, Han J, Ahn CH. A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA). Lab Chip 2012; 12(21): 4257–4262.
15. Vashist SK, Czilwik G, van Oordt T, von Stetten F, Zengerle R, Marion Schneider E, Luong JHT. One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30min. Anal Biochem. 2014; 456: 32–37.
16. Vashist SK, Czilwik G, Venkatesh AG. Elisa system and related methods. WIPO Patent Pub No WO/2014/198836; 2014.
17. Boonlert W, Lolekha PH, Kost GJ, Lolekha S. Comparison of the performance of point-of-care and device analyzers to hospital laboratory instruments. Point of Care 2003; 2(3): 172–178.

The authors
Sandeep Kumar Vashist*¹ PhD, John H.T. Luong² PhD
¹Vallo Med Health Care GmbH, Castrop-Rauxel, Germany
²Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department of Chemistry and Analytical, Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland

*Corresponding author
E-mail: sandeep.vashist@vallomed.com

Molecular testing is increasingly recognized for guiding patient management and development of new targeted therapies. Meanwhile, there is an increased demand to perform testing on smaller volumes of tissue. Recent literature reports the use of ultrasensitive techniques to detect DNA mutations and translocations from stained cytology smears [1].

by Dr Laleh Hakima, Dr Maja H. Oktay and Prof. Sumanta Goswami

Introduction
Mutational analyses are crucial for guiding treatment decisions. This is particularly important for targeted therapies with tyrosine kinase inhibitors (TKI) for lung non-small cell carcinoma (NSCC) and for surgical management of thyroid nodules with indeterminate cytological diagnoses. Both lung and thyroid lesions are frequently diagnosed using fine need aspiration (FNA). FNA is a preferred method of obtaining diagnostic samples compared to surgical excisions or core biopsies. The FNA procedure is minimally invasive, rapid, cost-effective and has reduced procedure-related complications.

Molecular diagnostic tests of cytological samples are often performed using cytology cell block preparations. However, insufficient cellularity of cell blocks is a frequent limitation to these tests. In addition, errors introduced by formalin fixation may also interfere with accurate detection of mutations. Recent studies have reported successful testing using cytology smears stained with Diff-Quik®, which are air dried and fixed in alcohol. This approach has several advantages compared to testing on paraffin-embedded tissue (PET). Alcohol is a great preservative for DNA and RNA. The sample quality is superior to PET because FNA samples are typically enriched for cancer cells and cytology smears yield intact whole nuclei rather than the nuclear fragments that are obtained by cutting PET [2]. Lastly, testing performed directly on microdissected tumour cells ensures that material is sufficient and representative of the tumour and results in a faster turnaround time. We used cytological direct smears stained with Diff-Quik® and Papanicolaou (Pap) to detect commonly encountered mutations in non-small cell lung carcinoma and thyroid carcinoma [1].

Lung- and thyroid-carcinoma mutations and fusion gene testing targets
Approximately 64% of all lung adenocarcinomas harbour somatic driver mutations. According to The Lung Cancer Mutation Consortium, the frequency of EGFR and KRAS mutations are 23% and 25%, respectively. The incidence of EML4–ALK translocation, mainly detected in non-smoker patients with wild-type EGFR and KRAS genes, is approximately 6% [2, 3]. Other less frequent mutations include: BRAF ~3%, PIK3CA ~3%, MET amplifications ~2%, ERBB2 (HER2/NEU) ~1%, MAP2K1 ~0.4%, and NRAS ~0.2% [2]. Additionally, new driver mutations have recently been identified in lung cancer patients [3]. The most common mutations in thyroid carcinoma involve BRAF, KRAS, and RET/PTC genes. The mutations in BRAF and KRAS are point mutations, whereas RET (PTC) mutations are gene rearrangements that result in fusion of the tyrosine kinase domain of the RET gene to various unrelated genes [4–7].

The EGFR mutation status of the cancer is associated with its responsiveness or resistance to EGFR TKI therapy. The EGFR gene is located on chromosome 7p11.2, spans about 200kb, and contains 28 exons. The gene encodes a protein of 464 amino acids. The EGFR protein is composed of an N-terminal extracellular ligand-binding domain, a transmembrane lipophilic segment, and a C-terminal intracellular region containing a tyrosine kinase domain. The EGFR tyrosine kinase modulates cell proliferation and survival. Activation of EGFR initiates signalling cascades involving several downstream pathways, including Ras GTPases, which induce crucial cellular responses, such as proliferation, differentiation, motility, and survival. EGFR mutations associated with objective responses to single-agent TKI therapy in lung adenocarcinomas are preferentially observed in females of East Asian ethnicity who are never smokers and have adenocarcinoma with lepidic growth pattern (formerly bronchioloalveolar carcinoma). In adenocarcinomas, the majority of mutations have been identified in exons 18–21 of the EGFR gene. These mutations can be roughly classified into three major categories: in-frame deletions in exon 19, insertion mutations in exon 20, and missense mutations in exons 18–21 [8].

The fusion of the echinoderm microtubule-associated protein-like 4 (EML4) gene to the anaplastic lymphoma kinase (ALK) gene, EML4–ALK, is the most common fusion and results from the joining of exons 1–13 of EML4 to exons 20–29 of ALK. At least seven EML4–ALK variants (V1–V7) have been identified in lung adenocarcinomas. All seven variants are formed through the fusion of the intracellular tyrosine kinase domain of ALK with a variably truncated EML4 gene promoter. Activated ALK is involved in the inhibition of apoptosis and the promotion of cellular proliferation through activation of downstream PI3K/AKT1- and MAPK1-signalling pathways. The key downstream effectors on the ALK pathway include the Ras-activated protein, mitogen-activated protein kinase 1 [MAPK1; also known as extracellular signal regulated kinase (ERK)], phosphatidylinositol 3-kinase (PI3K), and signal transducer and activator of transcription 3 (STAT3) signalling pathways. Ras/MAPKK1/MAPK1 pathways are critical for cell proliferation, whereas the PI3K/AKT1 and STAT3 pathways are important for cell survival. The histology of these tumours is typically characterized by mucin production and either a solid growth pattern containing signet ring cells in Western patients or an acinar growth pattern in Asian patients. Compared with patients with wild-type ALK and EGFR, patients with the EML4–ALK fusion gene tend to be younger, of Asian ethnicity, diagnosed at an advanced clinical stage at presentation, male dominant, and more likely to be never smokers, but with a comparable response rate to chemotherapy and overall survival. The EML4–ALK fusion gene is typically detected by fluorescence in-situ hybridization (FISH). It has been reported that although ALK-fusion-positive lung cancers are resistant to the EGFR TKIs, gefitinib, and erlotinib, they are sensitive to small molecule TKIs against ALK [8].

The BRAF^V600E point mutation involves nucleotide 1799 and results in a valine-to-glutamate substitution at residue 600 (V600E). B-Raf is a serine/theronine protein kinase involved in MAPK/ ERK signalling pathway and in regulation of cell proliferation and differentiation. It is found in approximately 40–45% of papillary thyroid carcinomas. Not all variants are equally affected; 60% of classic papillary, 80% of tall cell variant, and 10% of follicular variant harbour this mutation. Its detection is clinically significant because if represents a prognostic marker for thyroid papillary carcinoma, it is associated with extrathyroidal extension, advanced tumour stage at presentation, and lymph node or distant metastases. BRAF^V600E point mutation is also an independent predicator of treatment failure and tumour recurrence, even with patients with low-stage disease [4].

Case selection and molecular analysis techniques
Thirty-one cases of lung adenocarcinomas and 26 thyroid carcinomas (17 classic papillary, 7 follicular variant, and 2 follicular carcinomas) were selected from the archives. Molecular analysis was performed on PET and from either Pap or Diff-Quik^® stained smears for each case. The following mutations in lung adenocarcinomas were tested: EGFR point mutations in exons 20 and 21, in-frame deletions in exon 19; KRAS point mutations in codons 12, 13, and 61; and EML4–ALK translocation. Thyroid carcinomas were tested for the BRAF^V600E point mutation.

Smears were reviewed by two cytopathologists and areas containing at least 50 cancer cells without necrosis or inflammation were marked for analysis [9, 10]. Tumour cells from marked areas were microdissected using RNA/DNA co-purification solution (Zymo Technologies) [5, 6]. QClamp xenonucleic acid (XNA) technology was used to detect mutations in EGFR, KRAS and BRAF genes. The qClamp, a wild-type sequence-specific XNA probe, has a melting point higher than 72 °C and remains attached to the wild-type DNA during the PCR extension stage. If there is a mutation, the probe dissociates from the template, which allows amplification. The technology was optimized using wild-type DNA (Promega, Madison, WI) resulting in a DNA shift of more than seven cycle thresholds (CTs) with the XNA clamp. In samples with mutations, the shift was less than 5 C_Ts (Fig. 1).

Quantitative RT-PCR (RT-qPCR) was used to detect EML–ALK translocations. PCR primers were used to amplify both the 3ʹ and 5ʹ ends of the ALK transcript (Aanera Biotech). In the presence of the translocation, the transcription of the EML4–ALK fusion gene (under the control of a stronger EML promoter) resulted in higher than expected 3ʹ ends than 5ʹ ends which lead to lower CT values for the 3ʹ transcript. A difference of more than 10 C_Ts between 3ʹ and 5ʹ ends was considered positive (Fig. 1).

The sensitivity of our assay was determined using two lung cancer cell lines, H1975 and H2228 (ATCC), harbouring the L858R mutation in the EGFR gene and the EML4–ALK translocation, respectively. The mutation detection rate was 1%.

Results
Approximately 80.6% of lung cases had some form of molecular alteration detected. There was 100% concordance between PET and cytology smears. All cases tested positive by our laboratory were also positive by the reference laboratory. However, seven cases that tested negative for EGFR mutation by the reference laboratory were found to be positive in our lab. In other words, clamp qPCR methodology detected approximately 20% more EGFR mutations than the reference laboratory. Likewise, three cases that were negative for KRAS by the reference laboratory, tested positive in our laboratory. In addition, there were six cases (19%) that had more than one molecular alteration in our cohort. Five cases had two mutations (three had EGFR exon 19 and KRAS codon 12 mutations, two had KRAS codon 12 and EML4–ALK mutations) and one case had three mutations (EGFR exon 19 and 21 and KRAS codon 12 mutation). Although EGFR and KRAS mutations were previously thought to be mutually exclusive, our results confirm recent reports of simultaneous mutations detected in study samples [7]. Given the known heterogeneity of cancers this finding may be expected, but might not have been detected previously because most analyses are performed using standard-sensitivity techniques.

Of 26 thyroid cases, 18 (81%) were positive for BRAF^V600E mutation on both PET and cytology smears. Concordant results were obtained from both cytology smears and PET from cases tested by us and the reference laboratory. Both follicular carcinoma cases tested negative on cytology smears and PET for BRAF^V600E. The percentage of patients with a BRAF mutation detected by us was higher than expected from the literature (81% versus 50%) reflecting our ultrasensitive approach to mutation detection [10].

Conclusion
Our results indicate that qClamp technology and the RT-qPCR approach can be used successfully to detect most common targetable molecular alterations from cytology smears of lung and thyroid carcinomas. We report successful DNA and RNA isolation from both Diff-Quik^® and Pap stained smears. This methodology represents an ultrasensitive and accurate approach with a 1% mutation detection rate and a decreased turnaround time of 1 to 2 days. Such an ultrasensitive method for molecular testing is essential as smaller amounts of diagnostic material become available and targeted approaches will be aimed at both molecular alterations present in the majority of cells, and at those present in a minority of cells that potentially may represent a subpopulation susceptible to recurrence [11].

Abbreviations
Table 1 shows the gene symbols and the names and symbols of the proteins encoded by those genes.

References
1. Oktay MH, Adler E, Hakima L, Grunblatt E, Pieri E, Seymour A, Khader S, Cajigas A, Suhrland M, Goswami S. The application of molecular diagnostics to stained cytology smears. J Mol Diagn. 2016; 18(3): 407–415.
2. Tsao MS, Sakurada A, Cutz JC, Zhu CQ, Kamel-Reid S, Squire J, Lorimer I, Zhang T, Liu N, Daneshmand M, Marrano P, da Cunha Santos G, Lagarde A, Richardson F, Seymour L, Whitehead M, Ding K, Pater J, Shepherd FA. Erlotinib in lung cancer – molecular and clinical predictors of outcome. N Engl J Med. 2005; 353: 133–144.
3. Campbell JD, Alexandrov A, Kim J, Wala J, Berger AH, Pedamallu CS, Shukla SA, Guo G, Brooks AN, Murray BA, Imielinski M, Hu X, Ling S, Akbani R, Rosenberg M, Cibulskis C, Ramachandran A, Collisson EA, Kwiatkowski. DJ, Lawrence MS, Weinstein JN, Verhaak RG, Wu CJ, Hammerman PS, Cherniack AD, Getz G, Cancer Genome Atlas Research Network, Artyomov MN, Schreiber R, Govindan R, Meyerson M. Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat Genet. 2016; 48(6): 607–616.
4. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med. 2011; 135(5): 569–577.
5. Gupta N, Dasyam AK, Carty SE, Nikiforova MN, Ohori NP, Armstrong M, Yip L, LeBeau SO, McCoy KL, Coyne C, Stang MT, Johnson J, Ferris RL, Seethala R, Nikiforov YE, Hodak SP. RAS mutations in thyroid FNA specimens are highly predictive of predominantly low-risk follicular-pattern cancers. J Clin Endocrinol Metab. 2013; 98(5): 914–922.
6. Nikiforov YE, Ohori NP, Hodak SP, Carty SE, LeBeau SO, Ferris RL, Yip L, Seethala RR, Tublin ME, Stang MT, Coyne C, Johnson JT, Stewart AF, Nikiforova MN. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab. 2011; 96(11): 3390–3397.
7. Nikiforov YE, Yip L, Nikiforova MN. New strategies in diagnosing cancer in thyroid nodules: impact of molecular markers. Clin Cancer Res. 2013; 19(9):2283–2288.
8. Cheng L, Alexander RE, Maclennan GT, Cummings OW, Montironi R, Lopez-Beltran A, Cramer HM, Davidson DD, Zhang S. Molecular pathology of lung cancer: key to personalized medicine. Mod Pathol. 2012; 25: 347–369.
9. Marchetti A, Milella M, Felicioni L, Cappuzzo F, Irtelli L, Del Grammastro M, Sciarrotta M, Malatesta S, Nuzzo C, Finocchiaro G, Perrucci B, Carlone D, Gelibter AJ, Ceribelli A, Mezzetti A, Iacobelli S, Cognetti F, Buttitta F. Clinical implications of KRAS mutations in lung cancer patients treated with tyrosine kinase inhibitors: an important role for mutations in minor clones. Neoplasia. 2009; 11: 1084–1092.
10. Russo M, Malandrino P, Nicolosi ML, Manusia M, Marturano I, Trovato MA, Pellegriti G, Frasca F, Vigneri R. The BRAF (V600E) mutation influences the short and medium-term outcomes of classic papillary thyroid cancer, but is not an independent predictor of unfavorable outcome. Thyroid 2014; 24: 1267–1274.
11. Pon JR, Marra MA. Driver and passenger mutations in cancer. Annu Rev Pathol. 2015; 10: 25–50.

The authors
Laleh Hakima¹ DO; Maja H. Oktay¹ MD, PhD; Sumanta Goswami*² PhD
¹Department of Cytopathology, Montefiore Medical Center Bronx, NY, USA
²Department of Biology, Yeshiva University, New York, NY, USA

*Corresponding author
E-mail: Goswami@yu.edu

Uromodulin kidney disease is a rare autosomal dominant kidney disease, characterized by hyperuricemia, gout and progressive kidney failure. Affected patients typically need renal replacement therapy in middle age. A considerable number of patients may reach end-stage kidney disease without a correct diagnosis, making improvements in diagnostic methods of vital importance.

by Dr Tamehito Onoe and Dr Mitsuhiro Kawano

Introduction
Uromodulin (UMOD), also known as Tamm–Horsfall protein, is the most abundant protein in healthy human urine. UMOD protein is a kidney-specific protein which is exclusively produced at the epithelial cells lining the thick ascending limb (TAL) of Henle’s loop.

The roles of urinary UMOD protein are assumed to be to protect against urinary tract infection, prevent urolithiasis formation and ensure water impermeability to create the countercurrent gradient. However, the accurate function and significance of UMOD protein are not yet fully elucidated [1].

Uromodulin kidney disease
Uromodulin kidney disease (UKD) is an inherited disease caused by UMOD gene mutations. So far, more than 100 mutations of the UMOD gene have been reported from all over the world [2]. Familial juvenile hyperuricemic nephropathy (FJHN), medullary cystic kidney disease type2 (MCKD2) and glomerulocystic kidney disease (GCKD), which are considered to be different diseases, have been proved to be caused by UMOD gene mutations [3]. Subsequently, because multiple names for one condition would be confusing and misleading, and also cysts are not pathognomonic for this disease, a new term, ‘Autosomal dominant tubulointerstitial kidney disease’ (ADTKD) was proposed in 2015 [4]. Mutations of renin (REN), hepatocyte nuclear factor 1β (HNF1β), and mucin-1 (MUC1) are also responsible for ADTKD besides UMOD. They all share common clinical characteristics, which are progressive kidney failure, tubulointerstitial nephritis and inheritance compatible with autosomal dominant trait with only trivial clinical differences. When UMOD mutation is identified in an ADTKD patient, the official diagnostic term is ADTKD-UMOD. However, UKD is also used to facilitate communication with patients, and so this term is used in the present article.

Patients with UKD have urinary concentration defect, hyperuricemia and gout from a young age. Their kidney function gradually deteriorates, and reaches end-stage kidney disease (ESKD) from 25 to 75 years of age. Their kidneys are usually of normal size or small and there are sometimes cysts, although the frequency of cysts does not differ from that of ‘non-cystic’ kidney diseases. Their urine tests usually show no or only very mild proteinuria or hematuria. The most prominent characteristic of UKD is a marked abundance of chronic kidney disease (CKD) patients in their pedigree, compatible with an autosomal dominant trait. Our group detected a novel A247P UMOD mutation in a UKD family (Fig. 1), many of whose members have hyperuricemia, CKD and ESKD, and are on hemodialysis (HD) therapy [5].

UKD is reported to be a rare disease, with a frequency of about 1.5 cases per million population. However, because hyperuricemia is a frequent complication in all CKD patients, when their family history is absent or unknown, it is difficult to suspect UKD, and so the frequency of UKD may be underestimated. This means that a certain proportion of UKD patients may reach ESKD without a correct diagnosis.

Diagnosis of uromodulin kidney disease
Clinically UKD should be suspected when a CKD patient has an abundant family history compatible with autosomal dominant trait, hyperuricemia, gout and bland urine findings. The final diagnosis of UKD is made by genetic test, which is, however, not commercially available, and only a limited number of laboratories are capable of performing it. So easier laboratory tests supportive of genetic tests would be helpful for the diagnosis of UKD and are awaited.

The renal histology of UKD patients shows nonspecific interstitial fibrosis, tubular atrophy and normal glomeruli. So it is difficult to make a diagnosis of UKD by ordinary histological methods. Moreover, not many UKD patients seem to undergo renal biopsy because their urine sediment shows no or only slight abnormalities and so clinicians may hesitate to undertake this invasive test. However, we believe that renal pathological examination is very informative not only for ruling out other kidney diseases but also for the diagnosis of UKD. UMOD proteins synthesized from mutated UMOD gene have protein folding disability and cannot escape from the endoplasmic reticulum (ER) of the epithelial cells. Immunostaining using anti-UMOD antibody in kidney sections of UKD patients shows massive UMOD accumulation in their epithelial cells (Fig. 2). Because of the question of whether there are any UKD patients among those who received kidney biopsy and were diagnosed as having nephrosclerosis or interstitial nephritis, we performed the following investigation.

In a 3787-sample kidney biopsy database of Kanazawa University, patients meeting all of the following criteria were selected for UMOD immunostaining. (1) Renal insufficiency (serum creatinine >1.0 mg/dL) below 50 years of age; (2) hyperuricemia: serum uric acid higher than 7mg/dl or under treatment for hyperuricemia; (3) no or only very mild abnormalities in urinalysis; and (4) no other apparent renal disease present clinically or histopathologically. Finally, 15 patients were selected and abnormal UMOD accumulations were detected in three independent patients by UMOD immunostaining. A247P UMOD gene mutations were detected in the proband of the family in Figure 1 and the other independent patient, indicating that they may share the same ancestor. The other patient had no family history of CKD. These results show that there may be more UKD patients than expected before, and also indicate that when kidney biopsy shows only nonspecific interstitial fibrosis in patients with renal insufficiency, UMOD immunostaining may be considered to detect UKD with or without a family history of CKD, especially with hyperuricemia and bland urinary findings.

Most of the synthesized UMOD protein is carried to the apical membrane of epithelial cells and excreted in the urine. However, a low but considerable amount of UMOD protein goes to the basolateral membrane and is secreted into the serum [6]. Serum UMOD protein concentrations are reported to be 45–490 ng/mL, while urine UMOD protein concentrations are 1000–80 000 ng/mL. The functions and significance of serum UMOD proteins are unknown.

Some results of animal experiments indicate that UMOD protein has a renoprotective effect against various types of injury. A renal ischemia-reperfusion experiment in UMOD knockout mice showed significantly worse results than in wild-type animals [7]. It is well known that urinary UMOD concentrations in UKD patients are decreased. The authors recently reported that serum UMOD protein concentrations are also significantly decreased in UKD patients besides urinary UMOD (Fig. 3). Serum and urinary UMOD concentrations decline in parallel with the decrease of estimated glomerular filtration rate (eGFR) due to the diminishment of UMOD producing epithelial cells in CKD patients. In UKD patients, the serum and urine UMOD concentrations were significantly lower compared with CKD patients beyond their eGFRs. Decreased serum and urinary UMOD concentrations may be good clues to suspect and diagnose UKD; however, verification in more UKD patients with various mutations will be indispensable.

Conclusions
So far no treatment has been devised that slows the rate of renal functional deterioration of UKD. At present, management for UKD patients is not different from that for other CKD patients. Anti-hyperuricemia drugs or anti-hypertensive therapy is used when necessary and the appropriate renal replacement therapy or renal transplantation should be considered when ESKD is reached. To clarify the pathogenesis and achieve effective treatment for UKD, establishment of more efficient diagnostic methods for UKD is expected. UMOD immunostaining for renal sections and measurement of serum and urinary UMOD concentrations are considered to be good modalities for the diagnosis of UKD. It is expected that through these tests, more UKD patients will be diagnosed at earlier stages and will be able to benefit from starting appropriate therapy before ESKD.

Recently some particular SNPs of UMOD promoter areas have been proved to be associated with hypertension or renal insufficiency from the genome-wide association study [8]. UMOD will likely attract greater attention as a renal-prognostic marker for not only UKD patients but also the general population.

References
1. Lhotta K, Piret SE, Kramar R, Thakker RV, Sunder-Plassmann G, Kotanko P. Epidemiology of uromodulin-associated kidney disease – results from a nation-wide survey. Nephron Extra 2012; 2: 147–158.
2. Scolari F, Izzi C, Ghiggeri GM. Uromodulin: from monogenic to multifactorial diseases. Nephrol Dial Transplant. 2015; 30: 1250–1256.
3. Hart TC, Gorry MC, Hart PS, Woodard AS, Shihabi Z, Sandhu J, Shirts B, Xu L, Zhu H, Barmada MM, Bleyer AJ. Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy. J Med Genet. 2002; 39: 882–892.
4. Eckardt KU, Alper SL, Antignac C, Bleyer AJ, Chauveau D, Dahan K, Deltas C, Hosking A, Kmoch S, Rampoldi L, Wiesener M, Wolf MT, Devuyst O. Autosomal dominant tubulointerstitial kidney disease: diagnosis, classification, and management-A KDIGO consensus report. Kidney Int. 2015; 88(4): 676–683.
5. Onoe T, Yamada K, Mizushima I, Ito K, Kawakami T, Daimon S, Muramoto H, Konoshita T, Yamagishi M, Kawano M. Hints to the diagnosis of uromodulin kidney disease. Clin Kidney J. 2016; 9: 69–75.
6. Bachmann S, Koeppen-Hagemann I, Kriz W. Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry. Histochemistry 1985; 83: 531–538.
7. El-Achkar TM, Wu XR, Rauchman M, McCracken R, Kiefer S, Dagher PC. Tamm-Horsfall protein protects the kidney from ischemic injury by decreasing inflammation and altering TLR4 expression. Am J Physiol Renal Physiol. 2008; 295: F534–544.
8. Trudu M, Janas S, Lanzani C, Debaix H, Schaeffer C, Ikehata M, Citterio L, Demaretz S, Trevisani F, Ristagno G, Glaudemans B, Laghmani K, Dell’Antonio G, Loffing J, Rastaldi MP, Manunta P, Devuyst O, Rampoldi L. Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression. Nat Med. 2013; 19: 1655–1660.

The authors
Tamehito Onoe MD, PhD and Mitsuhiro Kawano* MD, PhD
Division of Rheumatology,
Department of Internal Medicine,
Kanazawa University Hospital,
Kanazawa, 920-8641,
Japan

*Corresponding author
E-mail: sk33166@gmail.com

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 tumours, 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]. UriSed Technology is a unique solution in the market for the automation of sediment analysis, making traditional manual microscopy automatic [2]. The UriSed analysers provide a reliable and reproducible solution since 2007 [3]. As a new category instrument based on the improved UriSed Technology, the semi-automated UriSed mini was introduced in the market in 2015. In the present study, we evaluated the analytical performance of UriSed mini Semi-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 mini provides quantitative Red Blood Cell (RBC) and White Blood Cell (WBC) results, and semi-quantitative results for all other particle types: 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].
The instrument throughput is up to 60 samples per hour. Preparation of the UriSed mini analyser for measurement takes only some minutes, the only consumables are the patented disposable cuvettes for sample investigation and a manual pipette with appropriate pipette tip. 175 µl of urine sample is dispensed manually into the cuvette, further steps of the measurement sequence are completely automatic: 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 15 independent images at different positions of the sediment layer. These whole viewfield images are evaluated by a neural-network based image processing software.

Material and methods
Analysis of 311 samples was performed to evaluate UriSed mini 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 due to the single-use disposables. UriSed mini 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 mini was 4-24% while 5,5-67% in the case of manual microscopy [8]. Good correlation can be found between UriSed mini and manual counting chamber for formed elements. The Pearson correlation of quantitative parameters are 0.97 (RBC), 0.95 (WBC). The clinical evaluation of UriSed mini was based on McNemar test and concordance study. The results are shown in the following table.

Conclusion
The new UriSed mini utilizes the traditional gold standard method while eliminating the most time-consuming and operator-dependent procedures in laboratories performing manual microscopy. The semi-automated measurement process is reproducible and operator-independent. The UriSed mini semi-automated microscopy analyser requires manual sample pipetting, which makes the instrument small and simple to use. UriSed mini is a highly effective tool in a wide range of medical and clinical settings such as hospitals, clinics, accident and emergency departments and outpatient laboratories. In addition it can also serve as a backup instrument of automated sediment analysers.

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.

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

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