The identification of cell-free fetal DNA (cffDNA) in maternal plasma has led to the development of non-invasive prenatal testing (NIPT) for fetal aneuploidies risk assessment. The high accuracy of NIPT has profoundly influenced the field of prenatal care.

by F. Gerundino, Dr C. Giachini, C. Giuliani, E. Contini and Dr E. Pelo

Background
Several well-established screening approaches to estimate the personal risk for common autosomal aneuploidies such as trisomy 21 (T21, Down syndrome), trisomy 18 (T18, Edwards syndrome) and trisomy 13 (T13, Patau syndrome) are part of the standard prenatal care in many countries. These approaches are based on the combination of different parameters such as maternal age, markers in maternal serum and ultrasound findings in the first or second trimester of pregnancy. Overall, conventional screening tests show a detection rate (DR) of 80–95% with a high false positive rate (FPR) (3–5%). Pregnancies identified to be at high risk (using locally established cut-off values) are offered invasive prenatal diagnosis (IPD) to provide a definitive result. IPD, carried out using either chorionic villus sampling (CVS) or amniocentesis to obtain fetal cells, is associated with an estimated miscarriage risk of 0.5–1% [1]. Given the FPR of conventional screening protocols a not negligible number of pregnancies undergo unnecessary IPD. The identification of cell-free fetal DNA (cffDNA) in plasma of pregnant women [2] has opened new possibilities to improve non-invasive prenatal screening of common fetal aneuploidies. In the last decade, several groups developed massively parallel sequencing (MPS) – using targeted or whole genome approaches – of cell-free DNA (cfDNA) from maternal plasma to detect fetal aneuploidies [3, 4]. These approaches, referred to as non-invasive prenatal testing (NIPT) or non-invasive prenatal screening (NIPS), have been shown to outperform traditional screening protocols. According to a recent meta-analysis, the DR of NIPT was 99.2% for T21, 96.3%, 91.0% and 90.3% for T18, T13 and monosomy X, respectively; the FPR was below 1% for all of these aneuploidies [5]. Since 2011, NIPT became commercially available in the USA and China and was rapidly introduced into standard prenatal care in many countries.

Cell-free-DNA-based screening: validation of a method for fetal aneuploidies risk
The conventional first-trimester screening (FTS) is currently offered to all pregnant women by the public health system in Tuscany. Recently, we validated a NIPT method based on whole genome MPS approach [6], in order to introduce a more robust screening test within the public health system. In whole genome approach, maternal and fetal DNA fragments (called reads) are sequenced simultaneously in a single run. Sequence reads were aligned to specific chromosome locations within the human genome and the number of reads mapped to the chromosome of interest are counted. A relative increase or decrease in the number of reads respect to a predefined threshold value (Z-score) reveals a potential risk of aneuploidy for a specific chromosome. In particular, a trisomy was called when Z-score >3 (Fig. 1). MPS was performed on a total of 381 cfDNA samples isolated from maternal plasma by two steps: a first set of 186 euploid samples was analysed to generate a preliminary reference dataset (group A) and a second set of 195 samples (group B) – enriched by 69 aneuploid cases – was analysed in blind versus the reference dataset to verify the reliability of our sequencing protocol as well as the analysis method. One hundred and fifty samples from group A (80.6%) and 177 samples from group B (90.8%) gave resulted suitable (>10×106 mapped reads) for downstream data analysis. The two groups (A and B) were then merged to generate a definitive dataset (n=327), which was then used to re-analyse the whole study population. Since the fetal fraction (FF) (i.e. the proportion of fetal DNA to the total cfDNA in maternal plasma) is a parameter that strictly influences NIPT performance [7], a droplet digital PCR (ddPCR) protocol has been validated for its assessment [8]. FF quantification by ddPCR was performed in 178/381 (46.7%) samples after methylation-sensitive DNA digestion. Absolute quantification of both fetal (on digested RASSF1A) and total DNA (on TERT and undigested beta-actin/RASSF1A) was calculated as the ratio between the average copies/µL of fetal DNA and total DNA. An SRY assay was used for fetal gender assessment [6].

Results of the validation study
Considering the performance of the definitive reference dataset, all positive samples for T21 (n=43), T18 (n=6) and T13 (n=7) were correctly identified (sensitivity 99.9%). Five false positive (FP) results were observed: three for T21 (specificity 98.9%) and two for T13 (specificity 99.4%).

Z-score values of true positive (TP) cases for T21 and T13 were always higher than 4.6 and 6.6, respectively. Conversely, all Z-score values of FP cases for T21 and T13 lay within 3.0 and 4.0 (the so-called Z-score ‘grey zone’). Sex chromosome status was correctly assigned in 317/324 (97.8%) cases: 166 males, 149 females and 2 cases with monosomy X. In 3/327 (0.9%) samples fetal gender could not be assigned because of an inconclusive result in data analysis. Seven discordant cases between MPS and follow-up data were observed. The only case of false negative (FN) male has been explained by a low FF (0.3%), underling the importance of FF determination. Only two out of four cases with monosomy X were correctly identified by NIPT, while the remaining two cases were erroneously classified as male.

Discussion
NIPT is an accurate screening test without associated risk for the mother and/or the fetus and it can be performed early in pregnancies, starting from 9–10 weeks of gestation. It is suitable both in low- and high-risk pregnancies, even if the positive predictive value (PPV) of the test (the chance that the positive result is a true positive) is lower in low risk cohorts. Two large studies show that in the general population NIPT outperforms conventional screening tests for T21 with a PPV ranging from 45.5 to 80.9% versus a PPV of 3.4–4.2% [9, 10]. Pre- and post-test counselling to inform patients about benefits, risks, test failure and testing alternatives should be provided before offering cfDNA screening. Owing to a series of intrinsic limitations, NIPT cannot be considered a diagnostic tool, despite its high performance. In the management of pregnancies with a high-risk NIPT, the possibility of FP results should always be taken into account and IPD should be recommended after a NIPT-positive result. cffDNA derives from the apoptosis of the placental cytotrophoblast cells, therefore in rare cases it may not represent the genetic constitution of the fetus. FP results may arise from confined placental mosaicism (CMP) in which some or all trophoblastic cells are trisomic, whereas the fetus is normal (1–2% of first-trimester placentas) [11]. FN results are a very rare occurrence and can be explained by fetoplacental discrepancies, in which the fetus shows an abnormal karyotype but the chromosome aberration is absent in the cytotrophoblast and, therefore, in the cffDNA. Additional sources of FP results can be unanticipated finding such as maternal chromosome abnormalities (maternal mosaicisms, microdeletions and other copy-number variations) or maternal malignancy, or the presence of a vanishing twin with an early loss of a trisomic fetus.

Failure to provide a result occurs in 1.6–6.4% of NIPT. Both laboratory technical issues or low FF can cause the failure [12]. Low FF in frequently found in overweight pregnant women, in which the low FF could be due to a dilution effect of an increased blood volume or to the high turn-over of adipocytes [13]. In these cases it is not advisable to repeat the test on a new sample because the probability of a second test failure is quite high. An accurate clinical management of cases with low FF and normal maternal weight is instead recommended, because FF is lower in pregnancies with aneuploid fetuses (T18, T13, monosomy X and triploidy) compared to euploid pregnancies [14].

NIPT has rapidly spread in many countries through commercial provider, leading to a re-examination of current screening methods, and several models of implementation of NIPT have been proposed with pros and cons. Current guidelines recommend that “in countries where prenatal screening is offered as a public health service, governments and public health authorities should assume an active role to ensure the responsible introduction of NIPT” [15].

Our study represents the first experience of NIPT within the Italian public health system. Following our validation study, NIPT testing for T21, T13 and T18 has been introduced as a clinical service for all pregnant women after 10+4 week of gestation upon payment. Our regional health system has planned a pilot study of two years to evaluate the benefit-to-cost ratio of NIPT introduction into routine prenatal care to support the current screening strategy based on nuchal translucency measurement and maternal serum biomarker quantification. NIPT will be offered in an adequate context of pre- and post-test counselling as an alternative option to IPD in pregnant women with high risk after FTS and applying the national cut-off of 1:250. We expect that this strategy would lead to a significant reduction in unnecessary IPD due to FP results of FTS with a reduction in fetal losses associated to diagnostic procedures among high-risk women, allowing us to offer the best screening strategy currently available.

References
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The authors
Francesca Gerundino BS, Claudia Giachini PhD, Costanza Giuliani BS, Elisa Contini BS, Elisabetta Pelo* MD
Diagnostic Genetic Unit, Careggi University Hospital, Florence, Italy

*Corresponding author
E-mail: peloe@aou-careggi.toscana.it

Appropriate reference intervals are critical for interpretation of laboratory test results and accurate assessment of health and disease. However, pediatric reference intervals are severely lacking, leading to significant risk of misdiagnosis. CALIPER has addressed these gaps by establishing a robust reference interval database based on thousands of healthy children and adolescents.

by Victoria Higgins and Dr Khosrow Adeli

Introduction
The clinical laboratory provides objective data through laboratory testing of bodily fluids (e.g. serum, plasma) to aid in several aspects of medical decision making, including identifying risk factors and symptoms, diagnosing disease, and monitoring treatment. To correctly interpret laboratory test results, they are often compared to reference intervals (RIs), sometimes referred to as ‘normative’ or ‘expected’ values. RIs are commonly defined as the central 95% of the distribution of laboratory test results expected in a healthy, reference population [1]. Laboratory values that fall outside the appropriate RI may be interpreted as abnormal, possibly indicating the need for additional medical follow-up and/or treatment [2]. Given their critical importance to healthcare it would be expected that accurate RIs, appropriate for the patient population, are used in clinical practice. However, this is unfortunately far from the truth.

Importance of pediatric reference intervals
It can be challenging and costly for individual laboratories to develop RIs for their specific patient population, due to the necessity of recruiting a sufficiently large number of healthy individuals [i.e. The Clinical Laboratory Standards Institute (CLSI) recommends 120 individuals per partition] [1]. This is particularly true for pediatrics, a population in which unique RIs are of high importance. To interpret pediatric test results, laboratories often use RIs that were established on an adult reference population. The use of adult RIs to interpret pediatric test results can lead to erroneous and inaccurate interpretation. This is highlighted in Figure 1, which depicts the concentration of alkaline phosphatase (ALP) throughout pediatric, adult and geriatric age. It is evident that the pediatric population has vastly unique normative ALP values. Unique analyte concentrations in pediatrics is also true for sex hormones, growth hormones and several other analytes [3–5]. Therefore, children should not be viewed as small adults in the context of medical practice, but require separate RIs (i.e. partitions) for different age and/or sex groups, in addition to neonates and premature babies [5].

Closing the gaps in pediatric reference intervals
The current CLSI guidelines, which are mostly focused on adult RIs, acknowledge the special challenges of establishing age- and sex-specific pediatric RIs and recommend development of new initiatives to address the current gaps. The quality of a RI critically depends on the selected reference population. Therefore, the direct method of establishing RIs, which involves recruiting healthy individuals and applying exclusion criteria to select an appropriate reference population, is recommended over the indirect method, which involves using an already existing database (e.g. laboratory information system) to calculate RIs [1]. It is imperative for RI initiatives to focus on recruiting a sufficiently large and healthy reference population to accurately establish appropriate RIs for the pediatric population (i.e. using the direct method). Recognizing the critical need to establish pediatric RIs, several national initiatives have collected health information and blood samples from healthy pediatric populations. These initiatives include KiGGS in Germany [6], the Lifestyle of Our Kids (LOOK) study in Australia [7], CHILDx in the United States [8–10], The COPENHAGEN Puberty Study in the Nordic countries [11], and The Canadian Laboratory Initiative on Pediatric RIs (CALIPER) in Canada [5, 12].

The KiGGS initiative collected comprehensive, nationwide data on the health status of over 17 000 children and adolescents aged 0 to 17 years, across 167 locations in Germany [6]. This study has focused on laboratory parameters of general health indices, markers of nutritional status, immunization status, iron metabolism, thyroid, and indices of atopic sensitization. They have published age-dependent percentiles (3rd to 97th) in German, which may serve as a basis for RIs [13]. The LOOK study in Australia developed age-specific RIs for 37 chemistries, immunoassays, and derived parameters [7]. The CHILDx study was initiated in 2002 at ARUP (Associated Regional and University Pathologists) Laboratories and established RIs for 35 markers for children aged 6 months to 6 years and 58 markers for children aged 7–17 years [8–10]. The Nordic countries have also successfully established pediatric RIs for 21 biochemical properties using samples from healthy children and adolescents aged 5–19 years collected from schools from 2006–2008 in the Copenhagen area in Denmark as part of The COPENHAGEN Puberty Study [11]. However, arguably the most successful initiative has been the CALIPER project in Canada.

CALIPER project
The CALIPER project was initiated by The Paediatric Focus Group of the Canadian Society of Clinical Chemists (CSCC) and primarily based at The Hospital for Sick Children in Toronto (ON, Canada). CALIPER has recruited over 9 000 healthy children and adolescents from schools and community centres to participate at blood collection clinics by completing a health questionnaire, body measurements and donating a blood sample. Using this biobank of healthy pediatric samples, CALIPER has established age-, sex- and, for some biomarkers, Tanner Stage-specific pediatric RIs for over 100 biomarkers including, common biochemical markers, protein markers, lipids and enzymes [12], specialty endocrine markers [14], fertility hormones [15], cancer biomarkers [16], vitamins [17], metabolic disease biomarkers [18], testosterone indices [19] and specialized biochemical markers [20, 21]. All RIs were established in accordance with CLSI guidelines, including sample size requirements, outlier removal, statistical method for partitioning, as well as RI and confidence interval calculations [1].

The majority of RIs were established using Abbott ARCHITECT assays, initially limiting the direct applicability of the CALIPER database to all Canadian laboratories. CALIPER subsequently performed a series of transference and verification studies to expand the CALIPER database to additional assays commonly used in clinical laboratories, including Beckman, Ortho, Roche and Siemens [22–25]. Again, CALIPER performed these studies in accordance with CLSI guidelines and, in fact, often exceeded the sample size and statistical criteria requirements [1, 26]. The comprehensive CALIPER pediatric RI database is available online (www.caliperproject.ca), as well as through a mobile application (CALIPERApp) available on iTunes and Google Play. These tools allow the CALIPER database to be easily accessible to laboratory professionals, physicians, parents and patients.

Continued improvement in pediatric laboratory test interpretation
While significant improvements have been made in pediatric laboratory test interpretation over the past decade, several gaps remain. First, RI data for neonates (including premature babies) and infants (age 0 to <1 year) remains a challenge, owing to difficulties accessing a healthy neonate and infant population. However, the limited neonatal and infantile reference data CALIPER has collected highlights the profound differences in the newborn period, necessitating accurate RIs for this age group. For example, Figure 2 shows the dynamic concentration of creatinine throughout the pediatric age range, particularly the elevated and highly variable levels in the first two weeks of life. A large-scale, comprehensive study aimed at recruiting healthy neonates and infants is required to fill this gap. CALIPER is currently initiating a study with the aim of establishing a complete RI database for neonates and infants, which will greatly improve neonatal healthcare for premature babies, newborns, and infants from primary to complex, tertiary care pediatric centres.

Secondly, the effect of ethnicity on biomarker concentration remains to be comprehensively examined. The International Federation of Clinical Chemistry (IFCC) recommends that every country establishes RIs [27]; however, most nations adopt RIs from studies predominately performed in Western countries based on primarily Caucasian populations without considerations of ethnic differences. Although the majority of biomarkers do not differ between individuals of different ethnic backgrounds, a preliminary examination of the influence of ethnicity in pediatrics by CALIPER has shown that some biomarkers do significantly differ among ethnic groups, including immunoglobulin G (IgG), transferrin, ferritin, and follicle-stimulating hormone (FSH) [12, 14, 15]. Another study examined the influence of ethnicity in adults and found that serum creatine kinase (CK) activity is significantly higher for those of African ancestry. As elevated CK activity is an indicator of statin-induced myopathy, elevated CK activity in those of African ancestry could result in inappropriate discontinuation of statin therapy if ethnic-specific RIs are not used [28]. Another recent study used data from the National Health and Nutrition Examination Survey (NHANES) to develop racial/ethnic-specific RIs among Asians, Blacks, Hispanics, and Whites [29]. CALIPER has initiated a new study to robustly determine the effect of ethnicity on the concentration of routine serum biomarkers by examining and comparing reference values in the four major Canadian ethnic groups (i.e. Caucasian, South Asian, East Asian, and Black).

Lastly, as clinical laboratories adopt their RIs from numerous different sources, including textbooks, manufacturer product inserts, expert opinions, or published literature, RIs in clinical practice may vary substantially between laboratories. A national survey performed in Australia by the Australian Association of Clinical Biochemists (AACB) Harmonisation Group highlights the extensive variation in adult RIs used in clinical practice, which greatly compromises the consistency and reliability of laboratory test result interpretation and patient care [30]. A recent Canadian RI study (manuscript submitted; Adeli K, et al. 2017) by the CSCC Harmonized RI (hRI) Working Group, further highlights the considerable variation in RIs across laboratories with a greater variation observed in pediatric RIs in current clinical use, even between clinical laboratories using the same instrument. These surveys highlight the critical need for harmonized RIs in clinical practice. Initiatives in the Nordic countries [31], UK [32], Australia [33] and Japan [34] have already established harmonized RIs for a number of laboratory tests primarily for adults, but also for pediatrics. The CSCC hRI Working Group is now also working towards Canada-wide RI harmonization.

Conclusion
Children cannot be viewed as small adults and indeed require pediatric-specific RIs appropriately partitioned by age and sex for accurate laboratory test result interpretation. Several national initiatives have begun to address these critical gaps over the past decade by establishing age-, sex- and Tanner Stage-specific RIs for several major analytical platforms. The CALIPER initiative in Canada has arguably been the most comprehensive study to date, with clinical laboratories in several countries globally implementing the CALIPER database into clinical practice. Despite the significant strides recently achieved, further research is warranted in several areas including the establishment of RIs specific to the neonatal and infantile period, ethnic-specific RI for a subset of laboratory markers, and RI harmonization. Collectively, the comprehensive reference database published by CALIPER and the emerging data from ongoing studies directly address the evidence gap in pediatric RIs and contribute to evidence-based interpretation of laboratory test results and enhanced diagnostic accuracy of laboratory biomarkers in current clinical practice.

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The authors
Victoria Higgins PhD candidate; Khosrow Adeli* PhD, FCACB, DABCC, FACB
CALIPER program, Pediatric Laboratory Medicine, The Hospital for Sick Children, University of Toronto, Toronto, ON M5G 1X8, Canada

*Corresponding author
E-mail: khosrow.adeli@sickkids.ca

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