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Featured Articles
The clinical chemistry laboratory in the diagnosis and management of testicular cancer
Cancer of the testicles, primarily the germ cells, is a highly treatable disease common to young men. This article describes how chemical biomarkers are central to the diagnosis, characterization, therapeutic monitoring, prognosis and long-term surveillance in patients with testicular cancer.
by Dr Angela Cooper and Dr Seán Costelloe
Incidence of testicular cancer
Testicular cancer (TC) is relatively rare, accounting for approximately 0.7% of all UK male cancers, with a worldwide incidence estimated as ~7 per 100 000 [1, 2]. Incidence of TC has noticeably increased in industrialized countries over the last few decades, particularly in white males of European descent, although the reasons for this remain unclear [2–5]. Amongst younger men aged between 15 and 49 years in the United Kingdom and the United States of America, TC is the most common type of cancer observed [2, 3, 6, 7].
Classification of TC
Approximately 95% of malignant TCs originate from primordial germ cells, also known as germ cell tumours (GCTs) [3, 7–9]. However, rarely these malignancies may arise from extragonadal primary sites such as the retroperitoneum, mediastinum or pineal gland [3–5, 8, 10]. Germ cell tumours classified as seminomas (~40%) are predominantly formed of uniform cell types, whereas non-seminomatous germ cell tumours (NSGCTs), also accounting for ~40% of GCTs, originate from multiple cell types such as embryonal carcinomas, teratomas, choriocarcinomas and yolk sac carcinomas. GCTs arising from mixed germ cells comprise the remaining 20%. The World Health Organization (WHO) classification system for testicular tumours (Table 1) define five basic GCT types based on histological examination:
- Seminomatous GCTs
- Non-seminomatous GCTs (NSGCTs)
- Embryonal cell carcinomas
- Yolk sac tumours
- Teratomas
- Choriocarcinoma
The vast majority of non-GCTs are sex cord-gonadal stromal tumours involving the Sertoli or Leydig cells of the testicles, and are often benign [8, 9, 11].
‘Burned-out’ GCTs, or spontaneous regression of a testicular GCT, is a very rare phenomenon occasionally observed in male patients presenting with metastatic malignancy with an absence of primary testicular tumour. Often, the only remaining evidence of malignancy are features such as homogeneous scarring, hemorrhage, intratubular calcification and testicular atrophy. This may be associated with choriocarcinomas or teratomas [5, 12].
Testicular GCTs exhibit very diverse histology and immunostaining profiles, and have varying clinical progression and prognosis outcomes as demonstrated by the numerous methods of GCT classification systems. It is outside the focus of this paper to consider histology or immunostaining used in the identification and differentiation of GCTs, as these topics has been extensively documented in other review articles.
Treatment and cure rates in TC
Advances in treatment strategies, such as the use of cisplatin therapies [13], careful staging at diagnosis, early intervention using multidisciplinary teams, rigorous surveillance follow-up, and salvage therapy, means that GCTs are highly curable. Currently, expected cure rates of 95% are observed in patients who receive a TC diagnosis, and cure rates of 80% in patients with a diagnosis of metastatic TC [3, 13].
Causes and presentation of TC
The causes of TC cancer are still unknown, although cryptochordism is the best-characterized risk factor associated with TC. Research has shown that timing of orchiopexy impacts on future risk of TC development, suggesting hormonal changes during puberty are strongly associated with TC etiology in males. However, prenatal risk factors, environmental exposures in adulthood, male infertility, certain genetic or congenital disorders such as Down’s syndrome, Klinefelter’s syndrome, human immunodeficiency virus infection and intersex patients have also been associated with an increased TC risk [3, 5, 7].
Presentation of TC is often a painless lump in the testis body, but due to a frequent lack of pain, medical opinion is frequently delayed. A testicular mass or swelling, or episodic diffuse pain may be observed. More rarely, metastatic symptoms such back pain arising from retroperitoneal lymph node involvement, or coughing, pain or hemoptysis due to lung metastasis may be reported [3, 7, 8].
Diagnosis and staging of TC
Clinical suspicion of TC, such as altered testicular shape or non-painful swelling, should prompt a full physical examination and patient history, imaging to include testicular and abdominal ultrasound, as well as chest X-ray [14]. If metastasis is suspected, chest, abdominal and brain computerized tomography (CT), and bone scintigraphy should be undertaken [9]. Final diagnosis and prognosis requires biopsy sampling for histology and immunostaining profiling as appropriate, and in the majority of cases, treatment options should be based on the histology results [10]. Biochemical analysis should include initial concentrations of serum tumour markers (STMs). Metabolic biochemistry, liver function tests and a full blood count should be undertaken to determine general organ function, and may demonstrate evidence of metastasis [9].
This collective information can be used to reference the Tumour-node-metastasis (TNM) Classification of Malignant Tumours staging system (Table 2). This cancer staging system is based on primary tumour site, nearby lymph node involvement, and presence of distal metastatic spread from initial primary tumour site [4, 15]. The use of STMs as a fourth staging system has added diagnostic and prognostic value, independent of the TNM system (Table 3) [9]. The decision for chemotherapy or radiotherapy treatment for non-surgical metastatic disease is based on CT and/or magnetic resonance imaging (MRI) results, and concentrations of STMs [4].
The majority of patients (~75%) presenting with a testicular mass are diagnosed at stage 1 [7, 8]. At this stage, treatment options are typically surgery with an excellent cure rate. For metastatic disease, combinations of surgery, chemotherapy or radiotherapy are required depending on cancer mass, location and distal lymph node involvement [13]. Greater than 80% of patients with metastatic GCTs are successfully treated and cured.
Treatment of TC
TC cells are extremely sensitive to chemotherapy [9, 10]. Specifically, the standard chemotherapy regime consists of 3 or 4 cycles of bleomycin, etoposide and cisplatin (BEP) chemotherapy, or etoposide and cisplatin (EP) chemotherapy every 21 days [8, 9]. Surgery may be considered to remove residual masses post-chemotherapy. Data suggests a higher relapse rate in patients with NSGCTs than seminomas following an initial chemotherapy regime. This relapse rate can be used to further classify patients into good, intermediate and poor prognostic groups, using a combination of STM concentrations and location of primary tumour or metastases. Around 50–99% of patients can still expect to survive [8].
Salvage therapy, often in combination with chemotherapy, is reserved for patients who have relapsed, or for patients where cancer progression continues after following a standard chemotherapy regime. High-dose chemotherapy with autologous bone marrow transplant is a controversial approach for patients with a poor prognosis, and where a standard chemotherapy regime and salvage therapy has been unsuccessful. Initial studies are encouraging but further trials are required. A small cohort of patients have been identified who suffer a late relapse, i.e. >2 years post-diagnosis but also potentially ≥10 years post-diagnosis. These patients are less responsive to chemotherapy, so are treated primarily with surgery. Unfortunately, less than half will remain disease-free following surgical intervention [8, 9]. Chemotherapy-induced side effects are governed by the dose and combination of drugs used. This has triggered more recent trials designed at maintaining a cure rate but with reduced associated chemotoxicity [8].
The use of serum tumour markers in TC
The discovery of serum and urine tumour markers and the advent of chemotherapy have significantly improved cancer staging, management and prognosis in patients with TC. The benefit of initial STMs is predominantly with regard to disease staging, whereas serial STMs are particularly useful for monitoring response to treatment after surgery, chemotherapy or radiation therapy. STMs are useful because they are often detectable well before clinical radiological detection in patients. Furthermore, concentrations can be helpful to differentiate GCT type. The detection of at least one elevated STM occurs in ~85% of NSGCTs, and the presence of elevated STMs occurs in significant numbers of pure seminoma cases [9, 10]. However, in rare cases where patients present with evidence of a testicular mass, radiographic evidence of metastatic disease, with significantly elevated alpha-fetoprotein (AFP) or human chorionic gonadotrophin (hCG) serum concentrations, it is advised that treatment is not delayed while awaiting histology results [10].
The American Society of Clinical Oncology recommend against using STMs as a screening test for GCTs in asymptomatic males. Given the low incidence and mortality of TC combined with the high cure rate, it is suggested a screening programme would be neither cost-effective nor decrease mortality [10]. Furthermore, although STMs can be helpful in combination with imaging techniques in the diagnosis of TC, normal STMs alone do not exclude TC and may also be raised in other conditions [3, 8–10]. Routine testicular examination via palpation is recommended in all males from puberty up to ~45 years. This is of particular importance for males with a past medical history that may suggest an increased GCT risk as detailed previously.
Commonly employed serum markers include: AFP and hCG as mentioned previously, hCG beta-subunit (hCGb), placental alkaline phosphatase (PLAP) and lactate dehydrogenase (LDH). Alpha-fetoprotein levels are elevated in teratocarcinoma or testicular embryonal carcinoma, while conversely, AFP is never elevated in pure seminomas. Human chorionic gonadotrophin elevations are associated with 10–15 % of pure seminomas. Lactate dehydrogenase is an enzyme found in all cell types, meaning it is less specific for TC, although it does have prognostic value in advanced stage GCTs [3, 9]. A decline in serial STM concentrations is useful to detect the presence of residual disease following surgery, or to assess response to chemotherapy. In both scenarios, the decline in STM concentrations should follow the half-lives of each marker [9].
There are detailed STM surveillance guidelines in place following surgery, which recommend a meticulous timetable of STM measurements and radiology imaging to detect disease recurrence depending on initial GCT type, thereby avoiding relapse and presentation at a later date with advanced stage disease [8, 9].
Future focus
While the majority of patients diagnosed with TC will survive, challenges still persist. Serum tumours markers have been pivotal to improved outcomes for patients with and without metastatic disease. Future research is focused on patients with an initial poorer prognosis, patients who have relapsed following first-line chemotherapy and patients who have a late relapse. Long-term health consequences for patients surviving TC, in particular side effects associated with chemotherapy and radiotherapy such as cardiovascular disease, impaired fertility and secondary cancers, continues to drive collaborative studies nationally and internationally to improve TC outcomes for the future.
References
1. Cancer registration statistics, first release, England, 2014. Office for National Statistics 2014. (http://web.ons.gov.uk/ons/rel/vsob1/cancer-statistics-registrations–england–series-mb1-/2014–first-release-/rpt-cancer-stats-registrations.html)
2. Hameed A, White B, Chinegwundoh F, Thwaini A, Pahuja A. A review in management of testicular cancer: single centre review. World J Oncol. 2011; 2: 94–101.
3. Bosl GJ, Motzer RJ. Testicular germ-cell cancer. N Engl J Med. 1997; 337: 242–254.
4. Bahrami A, Ro JY, Ayala AG. An overview of testicular germ cell tumors. Arch Pathol Lab Med. 2007; 131: 1267–1280.
5. Sesterhenn IA,Davis, CJ. Pathology of germ cell tumors of the testis. Cancer Control 2004; 11: 374–387.
6. Wu X, Groves FD, McLaughlin CC, Jemal A, Martin J, Chen, VW. Cancer incidence patterns among adolescents and young adults in the United States. Cancer Causes Control. 2005; 3: 309–320.
7. Hanna NH, Einhorn LH. Testicular cancer – discoveries and updates. N Engl J Med. 2014; 371: 2005–2016.
8. Horwich A, Nicol D,Huddart R. Testicular germ cell tumours. BMJ 2013; 347: f5526.
9. Barlow LJ, Badalato GM,McKiernan JM. Serum tumor markers in the evaluation of male germ cell tumours. Nat Rev Urol. 2010; 7: 610–617.
10. Gilligan TD, Hayes DF, Seidenfeld J, Temin S. ASCO clinical practice guideline on uses of serum tumor markers in adult males with germ cell tumors. J Clin Oncol. 2010; 6: 199–202.
11. Eble JN, Sauter G, Epstein JI, Sesterhenn IA. World Health Organization classification of tumours. Pathology and genetics of tumours of the urinary system and male genital organs. IARC 2004.
12. Ulbright TM. Germ cell tumours of the gonads: a selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Mod Pathol. 2005; 18: S61–S79.
13. Masters JR, Köberle B. Curing metastatic cancer: lessons from testicular germ-cell tumours. Nat Rev Cancer. 2003; 3:517–525.
14. Suspected cancer: recognition and referral guidelines [NG12]. National Institute for Health and Care Excellence (NICE) 2015. (https://www.nice.org.uk/guidance/NG12/chapter/1-Recommendations-organised-by-site-of-cancer)
15. Sobin LH, Gospodarowicz MK and Wittekind C. TNM classification of malignant tumours (7th ed). International Union against Cancer (UICC). Wiley-Blackwell 2009.
16. Albers P. (Chair), Albrecht W, Algaba F, Bokemeyer C, Cohn-Cedermark G, Fizazi K, Horwich A, Laguna MP, Nicolai N, Oldenburg J. Guidelines on testicular cancer. Eur Urol. 2015. (https://uroweb.org/guideline/testicular-cancer/)
The authors
Angela Cooper* PhD, Seán Costelloe, PhD
Derriford Combined Laboratory, Plymouth Hospital NHS Trust, Plymouth, UK
*Corresponding author
E-mail: angelacooper5@nhs.net
Inductively coupled plasma mass spectrometry: the future of sweat analysis?
Sweat chloride is the gold standard diagnostic test for cystic fibrosis (CF) offering direct measurement of cystic fibrosis transmembrane conductance regulator (CFTR) protein function. Current methods are labour-intensive, complex, time-consuming and require relatively large sample volumes. Inductively coupled plasma mass spectrometry (ICP-MS) is an emerging technology capable of providing rapid and accurate sweat chloride concentrations from small sample volumes.
by Dr Anna Robson, Dr Alexander Lawson and Dr Stephen George
Background
Cystic fibrosis (CF) is a life limiting inherited disorder with an incidence of 1 in 2500–3500 live births in the UK and USA [1]. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene translate to dysfunction of the CFTR protein, responsible for controlling transepithelial chloride transport. CF is a multisystem disease, due to the ubiquitous location of the CFTR, however, it is most commonly associated with pulmonary and pancreatic pathologies. Data from the Cystic Fibrosis Foundation Patient Registry (CFFPR) annual report showed that approximately 80% of CF patients are on pancreatic enzyme replacement therapy (PERT) and over 70% of CF mortality is a consequence of respiratory or cardiorespiratory related causes [2].
Implementation of newborn screening pathways for CF has enabled early diagnosis, with advancements in treatment strategies showing significant benefits to patients over the last 25 years. CFFPR data shows that patients now have a median predicted survival of 39.3 years and a higher proportion of adults than children, with CF, was reported in the USA for the first time in 2014 [2]. Patients’ quality of life is also improving with better lung function at the age of 18 years, more patients graduating from higher education and an increased number of viable pregnancies in female patients [2].
There are currently over 2000 known genetic mutations of the CFTR gene associated with CF, many of which can be categorized into five classes [3]. Classes I–III give rise to the most severe phenotypes due to absence or non-function of CFTR, whereas residual CFTR function is observed in patients with class IV–V mutations [2, 3]. Molecular genetic testing has become an increasingly important aid to the diagnostic pathway for CF, especially for patients with rarer mutations and milder forms of the disease. However, molecular analysis is insufficiently sensitive and specific enough to be used as a first line test as rare variants can be missed. Use of next generation sequencing is currently prohibited by cost but may become a more viable option in the future. The UK newborn screening program for CF utilizes a combination of immunoreactive trypsinogen (IRT) and genetic testing for the most common mutations. Although IRT is a good screening tool in neonates, it is not diagnostic and is unsuitable for use in adults. All babies that screen positive for CF are referred for diagnostic confirmation by sweat testing.
Diagnosis of CF
More than six decades since its inception in 1959 [4], quantification of chloride ions in sweat remains the gold standard diagnostic test for diagnosis of CF. In normal functioning sweat glands, isotonic sweat is secreted into the secretory coil. Sodium and chloride are then reabsorbed in the water-impermeable reabsorptive duct via the epithelial sodium channel (ENaC) and the CFTR respectively. Reabsorption of chloride is reduced in CF patients with defective or absent CFTR, thus resulting in sweat electrolyte loss. Sweat concentrations, therefore, provide a direct measurement of electrolyte secretion. Elevated sweat sodium levels are also observed in CF due to the dependence of ENaC activation on CFTR function.
Sweat chloride measurements demonstrate 98% diagnostic specificity for CF [5]. Research has also shown correlations between the type of genetic mutation and chloride concentration [2, 6]. Methods employed in sweat analysis include osmolality, conductivity and electrolyte concentration, however current guidelines developed by the UK Royal College of Paediatrics and Child Health (RCPCH) and the Association of Clinical Biochemistry (ACB) recommend sweat chloride as the analyte of choice for CF diagnosis [7]. Sweat conductivity measurements are accepted for screening purposes in patients over 6 months of age provided that all positive and borderline results are followed up with a chloride measurement [7]. Sweat sodium is no longer recommended for CF diagnosis as it is less reliable than chloride as an indicator of CFTR function [7]. Most laboratories providing sweat analysis measure a combination of analytes, typically chloride with either conductivity or sodium, using the latter two for quality control purposes only [5].
There are currently two accepted methods for collecting sweat following pilocarpine stimulation, the Gibson and Cooke technique (GCT) and the Wescor Macroduct® collection system (WMCS). Using the GCT, sweat is collected onto pre-weighed chloride-free filter paper and eluted in the laboratory for analysis. Sweat is collected into a plastic capillary in the WMCS closed system, thus reducing analytical errors associated with weighing, dilution and evaporation. A minimum sweat secretion rate of 1 g/m2/min is recommended to obtain an accurate chloride concentration. This equates to approximately 75 mg or 15 μL of sweat, depending on the collection method, in 30 minutes [1]. Low sweat rates indicate either suboptimal sweat secretion by the patient or sample evaporation, both of which can affect the accuracy of electrolyte measurements [5]. RCPCH/ACB guidelines therefore recommend duplicate analysis, on each sweat sample collected, to minimize analytical imprecision due to the manual nature of sweat testing [7]. Studies report conflicting data in relation to which collection system yields a higher insufficient rate for sweat analysis [8, 9]. However, the primary limitation of WMCS compared to GCT is reduced sample volume for analysis when a sufficient sweat rate has been achieved. A recent UK audit at Heart of England NHS Foundation Trust (HEFT) showed that more than 30% of samples with a sufficient sweat rate (>15 μL) were insufficient for duplicate ion selective electrode (ISE) analysis when using the WMCS.
Current methods of analysis
Currently accepted methods for sweat chloride quantification include coulometry, colourimetry, and ISE analysis, of which coulometry is the most commonly used (112/161 UK laboratories enrolled in the UKNEQAS external quality assurance (EQA) scheme). Sweat-Chek equipment is recommended for conductivity measurements and accepted methods for sodium include flame photometry, ISE and atomic absorption spectroscopy [6, 7]. All of these methods require manual measurement of each sample, thus occupying the time of a specially trained member of staff for the analysis duration. Dedicated instrumentation is often used for each analyte and sample volume requirements are relatively large compared to the minimum accepted collection using the WMCS. Sweat analysis is therefore complex and time-consuming. The need for dedicated instrumentation, specifically trained staff and laboratory time also carries a cost burden for NHS laboratories with increasing budget restrictions.
Inductively coupled plasma mass spectrometry
At HEFT, we have developed a method for the analysis of sweat sodium and chloride using inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS is an emerging technology in the clinical laboratory, primarily used for determining the elemental composition of samples and recently applied for sweat analysis [10, 11]. Benefits of ICP-MS include rapid and batched measurements, reproducibility, increased sensitivity and specificity compared to traditional methods, simultaneous quantification of multiple elements and ‘walk-away’ analysis. The ICP-MS method for sweat sodium and chloride uses a simple dilute and shoot approach, requiring just 2 μL of sample. The method was found to be both accurate and precise. Comparison studies using EQA samples showed a 3.6 % bias compared to target values [UKNEQAS EQA scheme all laboratory trimmed mean (ALTM)] (Fig. 1a); however, this was not statistically significant at clinical decision limits (30–60 mmol/L) and results were comparable to the coulometry method currently in use. Recommended acceptable precision (<5% CV), as defined by RCPCH/ACB guidelines, was obtained for all clinically relevant concentrations for quality control (QC) samples (Fig. 1b) [1, 7].
ICP-MS has numerous advantages, compared to coulometry, for sweat chloride analysis. Firstly, the low sample volume requirement allows for duplicate measurements on minimum viable samples (15 μL). Analysis run time is approximately 30–60 minutes depending on the number of samples, and ICP-MS is a ‘walk-away’ method. Staff are, therefore, available to carry out other work once the samples have been prepared and placed on the auto-sampler which is advantageous compared to current methods that can occupy a dedicated member of staff for up to half a day (Fig. 2). Improvements in laboratory efficiency are gained by moving away from dedicated chloride and conductivity meters to analysis using equipment already in use for the trace metal service. The main limitation of chloride analysis by ICP-MS at present is contamination due to the instrument tuning solution containing hydrochloric acid. Hence, care must be taken to ensure that the lines of the inlet system have been rinsed for long enough to remove any residual chloride before analysis. Clearly a tuning solution containing a different acid (e.g. nitric acid) would be beneficial and work is underway to source such a reagent. Overall, ICP-MS provides a much more efficient and cost-effective process for sweat analysis as illustrated in Figure 2.
Summary
Sweat chloride analysis is the gold standard test for diagnosis of CF; however, current methods are time-consuming, costly and require large sample volumes relative to the minimum acceptable collection. ICP-MS is a relatively new analysis platform in the clinical environment and is therefore not yet included in any guidelines. However, this technique presents an attractive alternative to current methods for rapid and accurate sweat analysis using small sample volumes. ICP-MS has the potential to benefit sweat testing, improving efficiency and reducing costs in the clinical laboratory.
Acknowledgements
The authors would like to acknowledge Dr Chris Chaloner PhD FRCPath and Lesley Tetlow FRCPath, Central Manchester University Hospitals NHS Foundation Trust, for their assistance in proofreading the manuscript.
References
1. Farrell PM, Rosenstein BJ, White TB, Accurso FJ, Castellani C, Cutting GR, Durie PR, Legrys VA, Massie J, et al. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report. J Pediatr. 2008; 153:S4–S14.
2. Cystic Fibrosis Foundation Patient Registry Annual Data Report 2014. (https://www.cff.org/2014-Annual-Data-Report.pdf)
3. Veit G, Avramescu RG, Chiang AN, Houck SA, Cai Z, Peters KW, Hong JS, Pollard HB, Guggino WB, et al. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol Biol Cell. 2016; 27(3): 424–433.
4. Gibson LE, Cooke RE. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Paediatrics 1959; 23(3): 545–549.
5. LeGrys VA. Sweat testing for the diagnosis of cystic fibrosis: Practical considerations. J Pediatr. 1996; 129: 892–897.
6. Mishra A, Greaves R, Massie J. The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era. Clin Biochem Rev. 2005; 26(4): 135–153.
7. Royal College of Paediatrics and Child Health. Guidelines for the performance of the sweat test for the investigation of cystic fibrosis in the UK, 2nd version. March 2014. (http://www.rcpch.ac.uk/system/files/protected/page/Sweat%20Guideline%20v3%20reformat_2.pdf)
8. Laguna TA, Lin N, Wang Q, Holme B, McNamara J, Regelmann WE. Comparison of quantitative sweat chloride methods after positive newborn screen for cystic fibrosis. Pediatr Pulmonol. 2012; 47: 736–742.
9. Hammond KB, Turcios NL, Gibson LE. Clinical evaluation of the macroduct sweat collection system and conductivity analyzer in the diagnosis of cystic fibrosis. J Paediatr. 1994; 124(2): 255–260.
10. Pullan NJ, Thurston V, Barber S. Evaluation of an inductively coupled plasma mass spectrometry method for the analysis of sweat chloride and sodium for use in the diagnosis of cystic fibrosis. Ann Clin Biochem. 2013; 50(Pt 3): 267–270.
11. Collie JT, Massie RJ, Jones OA, Morrison PD, Greaves RF. A candidate reference method using ICP-MS for sweat chloride quantification. Clin Chem Lab Med. 2016; 54(4): 561–567.
The authors
Anna Robson*1 PhD; Alexander Lawson2 PhD, FRCPath; Stephen George2 PhD, FRCPath
1Department of Clinical Biochemistry, Central Manchester University Hospitals NHS Foundation Trust, Newborn Screening Laboratory, Genetic Medicine, St. Mary’s Hospital, Oxford Rd, Manchester, UK
2Department of of Clinical Chemistry and Immunology, Birmingham Heartlands Hospital, Bordesley Green East, Birmingham, UK
*Corresponding author
E-mail: anna.robson@cmft.nhs.uk
Evaluation of UriSed mini semi-automated urine microscopy analyser
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
