AM KOPPS1, J MEIENBERG1, R BRUGGMANN2, K OEXLE1, G MATYAS1
1Center for Cardiovascular Genetics and Gene Diagnostics, Foundation for People with Rare Diseases, Schlieren-Zurich, Switzerland, 2Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of Berne, Berne, Switzerland
Current clinical next-generation sequencing makes use of gene panels and exome analysis, both of which involve selective capturing of target regions. However, capturing has limitations in sufficiently covering coding exons, especially GC-rich regions. We compared whole exome sequencing (WES, SureSelect Human All Exon v5+UTR) with the most recent PCR-free whole genome sequencing (WGS, Illumina TruSeq) for five female samples. We assessed the proportion of completely and sufficiently covered (313×) RefSeq coding exons. Thereby we analyzed (i) the entire exome and subsets of clinically relevant exons, i.e. (ii) genes recommended by the American College of Medical Genetics or (iii) exons with mutations recorded in the Human Gene Mutation Database. PCR-free WGS appears to be insensitive to GC content and is thus able to provide hitherto unprecedented complete coverage of the coding region of the genome. Although the average read depth was less than half (65× in WGS vs. 154× in WES), the proportion of completely and sufficiently covered coding exons was significantly higher in PCR-free WGS for all analyzed set of exons. The advantage of WGS does not only include the potential of identifying non-coding pathogenic variation but, in view of its more homogenous and complete exomic coverage, WGS is the better WES, thereby outweighing the higher costs. Thus, from a clinical/technical point of view capturing is no longer necessary for the most comprehensive genomic testing of Mendelian disorders.
1Departement of Laboratory Medicine (EOLAB), Ente Ospedaliero Cantonale, Lugano (Switzerland), 2Department of Gynecology and Obstetrics, Ente Ospedaliero Cantonale, Lugano (Switzerland), 3Centro di medicina di laboratorio Dr. Risch, Lugano (Switzerland), 4Departement of Nuclear Medicine and Thyroid Center, Oncology Institute of Southern Switerzland, Bellinzona (Switzerland)
Late onset hypogonadism (LOH) is a clinical and biochemical syndrome defined as the presence of three sexual symptoms (decreased frequency of morning erection, decreased frequency of sexual thoughts and erectile dysfunction) combined with a total testosterone (T) < 11 nmol/L. The etiology of LOH, is not completely understood and it is probably multifactorial.
The aim of this study was to provide age and method-specific normal ranges for total T in healthy men. We used a carefully selected group of 300 eugonadal men, with no known history of depression, diabetes, hypertension or erectile dysfunction, to measure total T. We compared reference intervals among four different assays platforms (Architect i 1000 SR-Abbot®), UniCel DxI800-Beckman Coulter®), Cobas 6000-Roche®, Immulite-Siemens® ) as well as the gold standard liquid chromatography/mass spectrometry (LC-MS 6490 Agilent Technologies®). Statistical analysis were performed with Analyse-it software. As expected, we found substantial differences in T level concentrations measured by different methods. Therefore we provided method-specific reference values. Surprisingly, all the calculated reference ranges included the cut-off of 11 nmol/L, which is currently adopted for the diagnosis of hypogonadism, and the lower limit was far below this level.
Consequently, a substantial proportion of healthy donors showed total T level below 11 nmol/L. International guidelines for the identification of LOH should consider the analytical variation among different platform. Waiting for standardization/harmonization of total testosterone assays, method-specific cut-off level must be provided and adopted to correctly identify LOH avoiding inappropriate testosterone substitution prescriptions.
1Kantonspital Glarus AG, Glarus, Switzerland, 2Risch Laboratory Group, Schaan, Principality of Liechtenstein
The formation of solid material of organic or inorganic origin in the urinary system (urolithiasis) is a rather common disease caused by various environmental or genetic factors (1). Once such crystal aggregates are isolated from patients detailed analysis of their composition is, beside conventional urinary analysis including microscopy and sediment characterization, the most important step towards differential diagnosis of the principle of causality.
State of the art technologies for “stone analysis” (2) are infrared spectroscopy (IRS) and powder x-ray diffraction (XRD). If using IRS the attenuated total reflectance (ATR) technology allows an accelerated workflow with less material needed since embedment of analyte material into a KBr matrix is not necessary. Both in conventional IRS and ATR-IRS spectral readouts are analyzed either manually or automatically by comparison with reference spectra since characteristic absorption line groups allow distinguishing the different crystal types. If using automated analysis systems the user relies on both the quality of the comparison algorithm and the reference collection – both provided by the manufacturer of both instrument and software.
In the course of a platform design and evaluation study we tested two ATR-IRS systems from independent manufacturers by measuring diagnosed patient samples (n = 51), proficiency testing samples (PTS, n = 18), and mixtures of pure crystalline materials (n = 64) including interference testing with water, urine and whole blood (n = 30). Whereas most of the PTS materials were identified at least partially by both platforms, patient sample analysis revealed the data base dependency of the analysis setup – both between the manufacturers platforms and within a vendor platform since the primary diagnosis of these materials was done with an instrument of one of the vendors. The analysis of mixtures showed the limitations of semi-quantitative (percentage) stone analysis with ATR-IRS – a pure qualitative composition description with the information which component is predominant seems to be more feasible.
Taken together it can be concluded, that software and database evaluation is a critical step in introducing IRS based stone analysis in a routine laboratory setting. In critical cases (e.g. inconsistent “hit list” entries) a second opinion – preferably with a different method (e.g. XDR) seems necessary to minimize diagnostic errors.
(1) Romero V, Akpinar H, Assimos DG. Rev Urol. 2010;12:e86-e96.
(2) Schubert G. Urol Res 2006;34:146-150.
1Risch Laboratory Group, Schaan, Principality of Liechtenstein
Screening efforts for drug abuse testing usually rely on immunological methods applied to urines samples. Antibodies raised for such assays are designed to have broad structural cross-section allowing coverage of whole analyte classes such as “benzodiazepins”, “amphetamins” or “opiates”. Beside the obvious advantage of this approach in clinical settings aiming for fast orientating results in emergency situations, this cross-reactivity can be interpreted as major drawback of this strategy, since confirmative testing by specific methods as LC-MS is mandatory. In this context we report on an isolated positive amphetamine result in immunological urinary drug testing. Investigation of the medical history of the case revealed, that the patient was under therapy with lisdexamfetamine, a CNS stimulant used in the treatment of attention deficit hyperactivity disorder (ADHD). This compound is a classical prodrug; upon enzymatic cleavage of a peptide bond dextroamphetamine is released in the blood. On the basis of MS2 / MS3 experiments using LC-IT-MS technology it was possible to unequivocally identify the drug in urine and serum specimen. Since also the prodrug – which has a short half-life time of less than one hour – was found and confirmed in both materials, an amphetamine drug abuse was made highly unlikely. This prodrug / drug pair means an additional analytical challenge in amphetamine screening efforts and – as already known from benzodiazepine or TCA measurements – do make the distinction between TDM and toxicology to become more blurred than ever.
M STAMOULI1, A SKLIRIS1, A MOURTZIKOU2, A POULIAKIS2, E MOURNIANAKIS1
11. Laboratoire de Biochimie, Hôpital Naval d’ Atènes, Atènes, Grèce, 22. Laboratoire de Cytopathologie diagnostique, Hôpital Universitaire Attikon, Atènes, Grèce
Introduction: L’hemoglobine anormale appelée hemoglobine S (HbS), resulte de la mutation d’une base du 6eme codon du gene β de la globine. Le but de cet travail est de presenter un cas de drépanocytose heterozygote composite HbS/PHHF.
Description du cas: Un homme de 18 ans, originaire de Grèce, candidat de formation en plongée sous-marine, bénéficie d’une numération-formule sanguine lors d’une visite médicale. L’hémogramme montre une anémie microcytaire avec une hémoglobine (Hb) à 11.7 g/dL (normal 13.5-18.0 g/dL), un volume globulaire moyen (VGM) à 79.5 fL (normal 80 à 100 fL) une teneur globulaire moyenne en hémoglobine (TGMH) à 24,6 pg (normal 28 à 34 pg) et d’importantes anomalies érythrocytaires (anisocytose, anisochromie, poikilocytose, microcytose, nombreuses cellules cibles). Les leucocytes et les plaquettes sont en nombre normal: leucocytes à 9600/mm3 (normal 4000 à 12000/mm3), plaquettes à 171000/mm3 (normal 150000 à 450000/mm3), formule leucocytaire normale, concentration serique en ferritine de 56 μg/L (normal 30 à 240 ng/mL). L’électrophorèse de l’hémoglobine en gel d’agarose en pH alcalin (Hydragel 15 Hemoglobin, Sebia, Norcross GA) réalisée sur l’automate Hydrasys 2, donne les résultats suivants: présence d’hémoglobine S (HbS) à un taux de 45.9%, d’hémoglobine F à un taux de 52.3 % et d’hémoglobine A2 à un taux de 1.8 %, absence d’hémoglobine A. Le test de falciformation du métabisulfite est positif. Les resultats ont etés confirmés par une électrophorèse en gel d’agarose à pH acide (Hydragel Acid Hemoglobin, Sebia, Norcross GA).
Discussion: L’HbS peut se polymériser dans certaines circonstances (hypoxie, déshydratation, acidose ou hyperthermie). La polymérisation intracellulaire de HbS est responsable de la symptomatologie particulière de cette anémie, dominée par les crises de falciformation. La drépanocytose avec un taux élevé d’HbF est de meilleur prognostic, parce que les taux d’HbF supérieurs à 10 % inhibent partiellement la falciformation. La prévalence de la drépanocytose heterozygote composite HbS/PHHF et de 1% des syndromes drépanocytaires.
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