Verifying measurements on Siemens Atellica® instruments using clinically acceptable analytical performance specifications.

Measurements on clinical chemistry analysers must be verified to demonstrate applicability to their intended clinical use. We verified the performance of measurements on the Siemens Atellica® Solution chemistry analysers against the clinically acceptable analytical performance specifications, CAAPS, including the component of intra-individual biological variation, CVI. The relative standard uncertainty of measurement, i.e. analytical variation, CVA, was estimated for six example measurands, haemoglobin A1c in whole blood (B-HbA1c), albumin in urine (U-Alb), and the following measurands in plasma: sodium (P-Na), pancreatic amylase (P-AmylP), low-density lipoprotein cholesterol (P-LDL-C), and creatinine (P-Crea). Experimental CVA was calculated from single-instrument imprecision using control samples, variation between measurements on parallel instruments, and estimation of bias with pooled patient specimens. Each obtained CVA was compared with previously developed CAAPS. The calculated CVA was 1.4% for B-HbA1c (CAAPS 1.9% for single diagnostic testing, CAAPS 2.0% for monitoring after duplicate tests; IFCC units), 10.9% for U-Alb (CAAPS 44.9%), 1.2% for P-Na (CAAPS 0.6%, after triplicate testing 1.5%), 8.2% for P-AmylP (CAAPS 22.9%). The CVA was 4.9% for P-LDL-C (CAAPS for cardiovascular risk stratification 4.9% after four replicates), and 4.2% for P-Crea (CAAPS 8.0%). Three of the six measurands fulfilled the estimated clinical need. Results from P-Na measurements indicate a general need for improving the P-Na assays for emergency patients. It is necessary to consider CVI when creating diagnostic targets for laboratory tests, as emphasised by the CAAPS estimates of B-HbA1c and P-LDL-C.

Lactate and ammonia measurements during cardiopulmonary exercise testing and its recovery phase-Consideration of age and sex in its interpretation.

BACKGROUND: Cardiopulmonary exercise testing with lactate and ammonia samples is used in the diagnostics of metabolic myopathies. As the effect of age and sex on the exercise lactate and ammonia levels are incompletely characterized for clinical associations, our aim was to assess the effects of these factors on healthy subjects to improve the test's interpretation. METHODS: Seventy-three subjects (34 men and 39 women; age < 35 years, n = 26, 35-50 years, n = 23 and >50 years, n = 24) performed cardiopulmonary exercise tests with venous blood gases, plasma lactate and ammonia analyses at rest, during exercise, and 2, 4, 6, 10, 20 and 30 min into recovery. RESULTS: The lactate (p = 0.021-0.044) and ammonia values (p = 0.002-0.038) differed between men and women measured during recovery and between three age groups point-by-point in maximal exercise and the recovery phase and also longitudinally, most notably between <35- and >50-year-groups (lactate p = <0.001-0.040, ammonia p = 0.002-0.03). In the linear model, the yearly reduction of lactate was maximally -0.119 mmol/L and that of ammonia -1.514 µmol/L. The yearly reduction of lactate was greater in women than in men (-0.131 vs.-0.099 2 min into recovery), but for ammonia, the results were not as clear. CONCLUSIONS: Plasma lactate and ammonia concentrations measured during cardiopulmonary exercise were lower in older age groups, and their yearly reduction was also influenced by sex. These data give new information on lactate and ammonia levels and the effect of aging on them during exercise and recovery and may help assess cardiopulmonary exercise testing results.

Clinical decision limits as criteria for setting analytical performance specifications for laboratory tests.

BACKGROUND: The biological (CVI), preanalytical (CVPRE), and analytical variation (CVA) are inherent to clinical laboratory testing and consequently, interpretation of clinical test results. METHODS: The sum of the CVI, CVPRE, and CVA, called diagnostic variation (CVD), was used to derive clinically acceptable analytical performance specifications (CAAPS) for clinical chemistry measurands. The reference change concept was applied to clinically significant differences (CD) between two measurements, with the formula CD = z*√2* CVD. CD for six measurands were sought from international guidelines. The CAAPS were calculated by subtracting variances of CVI and CVPRE from CVD. Modified formulae were applied to consider statistical power (1-ß) and repeated measurements. RESULTS: The obtained CAAPS were 44.9% for urine albumin, 0.6% for plasma sodium, 22.9% for plasma pancreatic amylase, and 8.0% for plasma creatinine (z = 3, α = 2.5%, 1-ß = 85%). For blood HbA1c and plasma low-density lipoprotein cholesterol, replicate measurements were necessary to reach CAAPS for patient monitoring. The derived CAAPS were compared with analytical performance specifications, APS, based on biological variation. CONCLUSIONS: The CAAPS models pose a new tool for assessing APS in a clinical laboratory. Their usability depends on the relevance of CD limits, required statistical power and the feasibility of repeated measurements.

Constitutive expression of Steroidogenic factor-1 (NR5A1) disrupts ovarian functions, fertility, and metabolic homeostasis in female mice.

Steroid hormones regulate various aspects of physiology, from reproductive functions to metabolic homeostasis. Steroidogenic factor-1 (NR5A1) plays a central role in the development of steroidogenic tissues and their ability to produce steroid hormones. Inactivation of Nr5a1 in the mouse results in a complete gonadal and adrenal agenesis, absence of gonadotropes in the pituitary and impaired development of ventromedial hypothalamus, which controls glucose and energy metabolism. In this study, we set out to examine the consequences of NR5A1 overexpression (NR5A1+) in the NR5A1-positive cell populations in female mice. Ovaries of NR5A1+ females presented defects such as multi-oocyte follicles and an accumulation of corpora lutea. These females were hyperandrogenic, had irregular estrous cycles with persistent metestrus and became prematurely infertile. Furthermore, the decline in fertility coincided with weight gain, increased adiposity, hypertriglyceridemia, hyperinsulinemia, and impaired glucose tolerance, indicating defects in metabolic functions. In summary, excess NR5A1 expression causes hyperandrogenism, disruption of ovarian functions, premature infertility, and disorders of metabolic homeostasis. This NR5A1 overexpression mouse provides a novel model for studying not only the molecular actions of NR5A1, but also the crosstalk between endocrine, reproductive, and metabolic systems.

Retinoblastoma protein represses E2F3 to maintain Sertoli cell quiescence in mouse testis.

Maintenance of the differentiated state and cell cycle exit in adult Sertoli cells depends on tumor suppressor retinoblastoma protein (RB, also known as RB1). We have previously shown that RB interacts with transcription factor E2F3 in the mouse testis. Here, we investigated how E2f3 contributes to adult Sertoli cell proliferation in a mouse model of Sertoli cell-specific knockout of Rb by crossing these mice with an E2f3 knockout mouse line. In the presence of intact RB, E2f3 was redundant in Sertoli cells. However, in the absence of RB, E2f3 is a key driver for cell cycle re-entry and loss of function in adult Sertoli cells. Knockout of E2f3 in Sertoli cells rescued the breakdown of Sertoli cell function associated with Rb loss, prevented proliferation of adult Sertoli cells and restored fertility of the mice. In summary, our results show that RB-mediated repression of E2F3 is critical for the maintenance of cell cycle exit and terminal differentiation in adult mouse Sertoli cells.

Loss of Glis3 causes dysregulation of retrotransposon silencing and germ cell demise in fetal mouse testis.

Fetal germ cell development is regulated by an elaborate combination of cell-extrinsic and cell-intrinsic signals. Here we identify a novel role for the Krüppel-like transcription factor Gli-Similar 3 (Glis3) in male germ cell development in the mouse embryos. Glis3 is expressed in male germ cells during the brief window of time prior to initiation of piRNA-dependent retrotransposon surveillance. Disruption of Glis3 function led to a widespread reduction in retrotransposon silencing factors, aberrant retrotransposon expression and pronounced germ cell loss. Experimental induction of precocious Glis3 expression in vivo before its normal expression resulted in premature expression of several piRNA pathway members, suggesting that GLIS3 is necessary for the activation of the retrotransposon silencing programs. Our findings reveal an unexpected role for GLIS3 in the development of male germ cells and point to a central role for GLIS3 in the control of retrotransposon silencing in the fetal germline.

At the Crossroads of Fate-Somatic Cell Lineage Specification in the Fetal Gonad.

The reproductive endocrine systems are vastly different between males and females. This sexual dimorphism of the endocrine milieu originates from sex-specific differentiation of the somatic cells in the gonads during fetal life. Most gonadal somatic cells arise from the adrenogonadal primordium. After separation of the adrenal and gonadal primordia, the gonadal somatic cells initiate sex-specific differentiation during gonadal sex determination with the specification of the supporting cell lineages: Sertoli cells in the testis vs granulosa cells in the ovary. The supporting cell lineages then facilitate the differentiation of the steroidogenic cell lineages, Leydig cells in the testis and theca cells in the ovary. Proper differentiation of these cell types defines the somatic cell environment that is essential for germ cell development, hormone production, and establishment of the reproductive tracts. Impairment of lineage specification and function of gonadal somatic cells can lead to disorders of sexual development (DSDs) in humans. Human DSDs and processes for gonadal development have been successfully modeled using genetically modified mouse models. In this review, we focus on the fate decision processes from the initial stage of formation of the adrenogonadal primordium in the embryo to the maintenance of the somatic cell identities in the gonads when they become fully differentiated in adulthood.

From the Cover: Teratogenic Effects of in Utero Exposure to Di-(2-Ethylhexyl)-Phthalate (DEHP) in B6:129S4 Mice.

Intrauterine exposure to phthalates is known to cause disorders of male reproductive function including androgen insufficiency, decreased fertility, and germ cell defects in rodents. In this study, we set out to investigate the effects of intrauterine exposure to di-(2-ethylhexyl)-phthalate (DEHP) on fetal development of the B6:129S4 mouse strain. Time-mated pregnant C57BL/6 dams were exposed to 0, 5, 250, or 500 mg/kg DEHP with corn oil as the vehicle via oral gavage from embryonic days (E)7 to 16. Survival and gross morphology of the pups were analyzed one day after the last treatment. Anogenital distance (AGD) and testicular cell functions were examined in male embryos to confirm the known effects of phthalate exposure. DEHP exposure significantly reduced the survival rate of fetuses in the 250 and 500 mg/kg dosage groups compared with the control and 5 mg/kg groups. Exposure to 250 and 500 mg/kg DEHP was teratogenic and induced exencephaly and limb malformations such as polydactyly in the B6:126S4 embryos. No gross malformations were observed in control or 5 mg/kg DEHP groups. In male embryos, exposure to both 5 and 250 mg/kg DEHP in utero was sufficient to induce the formation of multinucleated germ cells in the testes and widespread changes in mRNA expression of germ cell, interstitium and Sertoli cell-associated genes. These findings reveal that intrauterine DEHP exposure has a strong teratogenic, and lethal impact on the fetuses of B6:129S4 mouse strain.

CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures.

BACKGROUND: G protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to GαS stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells. RESULTS: CANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement. CONCLUSIONS: Here we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells.