Optimization of hemolysis, icterus and lipemia interference thresholds for 35 clinical chemistry assays.

OBJECTIVES: Interference of chemistry assays by hemolysis, icterus and lipemia (HIL) was investigated on the Abbott Alinity c system. We sought to empirically establish optimized HIL index thresholds for the purposes of reporting HIL interference in a hospital laboratory and advising clinicians on the interpretation of laboratory results in the presence of hemolysis, icterus or lipemia. METHODS: HIL index values measured by spectrophotometry were compared with concentrations of hemoglobin, bilirubin and Intralipid. HIL interference of 35 Abbott Alinity chemistry assays was subsequently investigated by pairwise comparison of test results in pooled serum or plasma with those in test preparations spiked with hemolysate, bilirubin or Intralipid. Data generated from the interference experiments were critically assessed according to assay-specific acceptance criteria adapted from multiple sources, and optimized thresholds for HIL indices were established. RESULTS: Correlations between HIL index values and their corresponding concentrations of hemoglobin, bilirubin and Intralipid were, in general, very good within the ranges of interferent concentrations tested. Hemolysis significantly affected 12 of 35 assays, whereas bilirubin and Intralipid interfered with four and three assays, respectively. Both the direction and magnitude of Intralipid interference with the direct bilirubin assay were dependent on the concentrations of the analyte. CONCLUSIONS: HIL interference of the Abbott Alinity clinical chemistry assays investigated in this study was not uncommon. At present, there are no universally accepted criteria for defining significant assay interference for clinical practice. In establishing acceptance criteria for defining assay interference, each assay should be assessed according to both analytical criteria and clinical relevance.

Association of Cord Blood Thyroid-Stimulating Hormone Levels with Maternal, Delivery and Infant Factors.

INTRODUCTION: This study examined maternal, delivery and infant factors associated with cord thyroid-stimulating hormone (TSH) concentrations in an Asian population. METHODS: The Growing Up in Singapore Towards healthy Outcomes (GUSTO) study is a mother-offspring birth cohort from 2 major hospitals in Singapore. Cord serum TSH was measured using the Abbott ARCHITECT TSH Chemiluminescent Microparticle Immunoassay and the ADVIA Centaur TSH-3 Immunoassay. After excluding infants with a maternal history of thyroid disease, screening cord TSH results from 604 infants were available for multivariable regression analysis in relation to the factors of interest. RESULTS: Babies born by vaginal delivery had significantly higher cord serum TSH concentrations than babies born by caesarean section. Cord serum TSH concentrations differed significantly by measurement method. There was no association of cord TSH concentrations with ethnicity, sex, birth weight, gestational age, maternal body mass index, gestational weight gain, gestational diabetes mellitus status and other maternal, delivery and infant factors studied. CONCLUSION: Interpretation of cord serum TSH results may need to take into account mode of delivery and measurement method.

Commutable whole blood reference materials for hemoglobin A1c validated on multiple clinical analyzers.

Background The measurement of hemoglobin A1c (HbA1c) is important for diagnosing diabetes mellitus as well as assessing glycemic control in diabetic patients. Commutable whole blood certified reference materials (CRMs) are needed in the measurement of HbA1c for method validation and/or as quality controls. Methods We developed three levels of hemolyzed whole blood CRMs for HbA1c. The certified values were determined using liquid chromatography-isotope dilution tandem mass spectrometry method (LC-IDMS/MS) where two "signature" hexapeptides of HbA1c and hemoglobin A0 (HbA0) were used as the calibration standards. The concentrations of the hexapeptide solutions were determined by amino acid analysis by the LC-IDMS/MS method using amino acid CRMs as the calibration standards. The commutability study was conducted by measuring 25 patient specimens and the whole blood CRMs by both LC-IDMS/MS method and various routine methods using six different clinical analyzers. Results The certified values were determined to be 35.1±2.0, 50.3±1.9 and 65.8±2.6 mmol/mol, respectively. These CRMs showed good commutability on five of the six clinical analyzers but showed poor commutability on one of the clinical analyzers that used similar method as two other analyzers where good commutability was observed. Conclusions With certified target values based on metrological traceability and good commutability on most of the clinical analyzers, the developed whole blood CRMs can be used for method validation or as quality control materials in the measurement of HbA1c. The commutability study results also underscored the need of commutability testing of clinical CRMs using various clinical analyzers.

Pragmatic and evidence-based approach to paediatric cerebrospinal fluid reference limits for white cell count and concentrations of total protein and glucose.

BACKGROUND: It is often impractical for each laboratory to establish its own paediatric reference intervals. This is particularly true for specimen types collected using invasive procedures, for example, cerebrospinal fluid (CSF). METHODS: Published CSF reference intervals for white cell count, and concentrations of total protein and glucose were reviewed by stakeholders in a paediatric hospital. Consensus reference intervals for the three CSF parameters were then subjected to verification using guidelines from the Clinical Laboratory Standards Institute and residual CSF specimens. RESULTS: Consensus paediatric reference intervals adapted from published studies with minor modifications were locally verified as follows. White cell count (x106 cells/L): 0-20 (<1 month); 0-10 (1-2 months); 0-5 (>2 months). Total protein (g/L): 0.3-1.2 (<1 month); 0.2-0.6 (1-3 months); 0.1-0.4 (>3 months). Glucose (mmol/L): 2.0-5.6 (<6 months); 2.4-4.3 (6 months or older).

Gestational age-specific reference intervals for serum thyroid hormone levels in a multi-ethnic population.

BACKGROUND: Thyroid disorders are common during pregnancy. To date, a limited number of studies have reported differences in serum thyroid hormone concentrations between different ethnic groups. We sought to establish gestational age-specific reference intervals for serum levels of thyroid hormones in a multi-ethnic population and investigate whether separate reference intervals should be used for different ethnic groups. METHODS: A total of 926 pregnant women from multiple ethnic groups attended four separate study visits spanning the three trimesters. Venous blood samples were taken at 9 to 14 weeks, 18 to 22 weeks, 28 to 32 weeks, and 34 to 39 weeks of gestation. Serum concentrations of thyroid-stimulating hormone (TSH), free thyroxine (T4), free triiodothyronine (T3), total T4, total T3, thyroid peroxidase antibody and thyroglobulin antibody were measured using Abbott Architect immunoassays. A total of 562 women with singleton pregnancies were found to be negative for both thyroid autoantibodies at all four study visits and thus included in the reference sample group for the establishment of reference intervals (2.5th to 97.5th percentiles). RESULTS: Reference intervals for serum thyroid hormones at 9-14 weeks of gestation derived from the combined group of pregnant women are as follows: TSH, 0.01-2.39 mIU/L; free T4, 11.4-19.5 pmol/L; free T3, 4.23-6.69 pmol/L; total T4, 77.8-182.4 nmol/L; total T3, 1.39-2.97 nmol/L. No differences in the five thyroid parameters' reference intervals are detectable among the ethnic groups except that at study visit 3 (28-32 weeks of gestation), the upper reference limit of total T3 in Malays (3.20 nmol/L; 90% CI, 2.99-3.76 nmol/L) is slightly higher than that in Chinese (2.86 nmol/L; 90% CI, 2.70-2.98 nmol/L). CONCLUSIONS: The findings from this study on a multi-ethnic cohort highlight the importance of establishing locally derived and gestational age-specific reference intervals for the five thyroid hormone parameters.

Transient Neonatal Zinc Deficiency Caused by a Novel Mutation in the SLC30A2 Gene.

This is a case report of a 4-month-old full-term, fully breastfed boy who presented with a persistent periorificial and groin rash associated with poor weight gain and irritability. His serum zinc level was low. The mother's breast milk zinc level was found to be low despite her serum zinc levels being normal, confirming the diagnosis of transient neonatal zinc deficiency. Mutational analysis revealed a novel mutation in the mother's SLC30A2 gene, which encodes a zinc transporter expressed in mammary gland epithelial cells.

LDLR gene synonymous mutation c.1813C>T results in mRNA splicing variation in a kindred with familial hypercholesterolaemia.

Familial hypercholesterolaemia, one of the most common inherited diseases in the general population, is associated with mutations in at least three different genes including the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and protein convertase subtilisin/kexin type 9 (PCSK9) genes. In this report, we describe an unclassified DNA variant (c.1813C>T; p.Leu605Leu) within exon 12 of the LDLR gene in a kindred in which familial hypercholesterolaemia is associated with c.1813C>T heterozygosity. In silico analysis suggested that c.1813C>T might affect splicing of the LDLR gene by creating a cryptic donor splice site, which was confirmed by RT-PCR coupled with cDNA sequencing, to result in the loss of 34 base pairs in the coding sequence. The latter truncated mRNA is predicted to generate a frameshift leading to a premature stop at codon 652 and early termination of the low density lipoprotein receptor polypeptide chain, and thus provides a molecular basis for the hypercholesterolaemic phenotype. This case report highlights the emerging utility of RNA studies for the molecular diagnosis of familial hypercholesterolaemia in patients with potential mRNA splicing variants.

The role of ω-3 fatty acid supplemented parenteral nutrition in critical illness in adults: a systematic review and meta-analysis.

OBJECTIVE: To determine whether the supplementation of parenteral nutrition with ω-3 fatty acids confers treatment benefits to critically ill adult patients. DATA SOURCE: We performed computerized searches for relevant articles from 1996 to June 2011 on MEDLINE, EMBASE, and the Cochrane register of controlled trials and abstracts of scientific meetings from 2005 to 2011. STUDY SELECTION: Randomized controlled trials of ω-3 fatty acid supplemented parenteral nutrition in critically ill adult patients admitted to the intensive therapy unit, given in addition to their routine care, compared with parenteral nutrition without ω-3 fatty acid supplementation. DATA SYNTHESIS: Five fully published trials and three trials published in abstract form with 391 participants have been included. Overall trial quality was poor. Mortality data were pooled from eight studies with 391 participants. No differences were found with a risk ratio for death of 0.83 (95% confidence interval 0.57, 1.20; p = 0.32). Data for infectious complications were available from five studies with 337 participants. No differences were found, with a risk ratio for infection of 0.78 (95% confidence interval 0.43, 1.41; p = 0.41). Data for intensive therapy unit and hospital length of stay were available from six and three studies with 305 and 117 participants, respectively. With respect to intensive therapy unit length of stay, no differences were observed with a mean difference of 0.57 days in favor of the ω-3 fatty acid group (95% confidence interval -5.05, 3.90; p = 0.80). A significant reduction in hospital length of stay of 9.49 days (95% confidence interval -16.51, -2.47; p = 0.008) was observed for those receiving ω-3 fatty acid supplemented parenteral nutrition, but results were strongly influenced by one small study. CONCLUSIONS: On the basis of this systematic review, it can be concluded that ω-3 fatty acid supplementation of parenteral nutrition does not improve mortality, infectious complications, and intensive therapy unit length of stay in comparison with standard parenteral nutrition. Although ω-3 fatty acids appear to reduce hospital length of stay, the poor methodology of the included studies and the absence of other outcome improvements mean they cannot be presently recommended.

Statins and their interactions with other lipid-modifying medications: safety issues in the elderly.

Inhibitors of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, commonly known as statins, are widely used in both primary and secondary prevention of occlusive cardiovascular disease. Statins are effective not only in improving total and low-density lipoprotein cholesterol concentrations in blood but also in decreasing morbidity and mortality associated with cardiovascular diseases resulting from underlying atheroma. There is, however, evidence that statins are underutilized in elderly patients, possibly due to concerns about safety/tolerability issues or potential drug interactions, including interactions with other lipid-modifying medications, or both. In this review, we summarize the major adverse events associated with statin use, with particular reference to the elderly patient, including factors which might increase the risk of adverse effects. Potential drug interactions between statins and other lipid-modifying medications including fibrates, ezetimibe, nicotinic acid, bile acid sequestrants and omega-3-acid ethyl esters (fish oils) are specifically discussed. Clinical management strategies to avoid these drug interactions are outlined.

Vitamin D deficiency is common and associated with metabolic risk factors in patients with polycystic ovary syndrome.

Both vitamin D deficiency and polycystic ovary syndrome (PCOS) are associated with aspects of metabolic syndrome, but it is unclear whether vitamin D deficiency contributes to the metabolic disturbances commonly found in women with PCOS. This study sought to investigate (1) the prevalence of vitamin D deficiency in PCOS women in Scotland and (2) the relationship between vitamin D status and metabolic risk factors. This was an observational study on 52 women (25 in PCOS group and 27 in control group). Serum 25-hydroxyvitamin D concentrations less than 25 nmol/L were classified as severe vitamin D deficiency and were found in 44.0% and 11.2% of subjects in the PCOS and control groups, respectively (P = .047). Among the PCOS subjects, 25-hydroxyvitamin D concentrations were negatively correlated with body mass index (P = .033), C-reactive protein (P = .027), and free androgen index (P = .025) and positively correlated with quantitative insulin sensitivity check index (P = .035), high-density lipoprotein cholesterol (HDL-C) (P = .033), and sex hormone binding globulin (P = .038). Associations of vitamin D deficiency with quantitative insulin sensitivity check index and HDL-C were independent of body mass index and waist-to-hip ratio. Vitamin D deficiency is highly prevalent in PCOS women in Scotland, and a larger proportion of PCOS patients than control women were found to be vitamin D deficient. We also demonstrate correlations of vitamin D status with insulin sensitivity, HDL-C, and C-reactive protein in PCOS patients, which support the increasing evidence that vitamin D deficiency is associated with multiple metabolic risk factors in PCOS women.

Late-onset male hypogonadism: clinical and laboratory evaluation.

Late-onset male hypogonadism (LOH) is a clinical and biochemical syndrome associated with advancing age and characterised by low serum testosterone concentrations. An understanding of the physiology of androgens in the ageing man is essential for the appropriate diagnosis of LOH. Clinical assessment of androgen status relevant to clinical biochemists and chemical pathologists is outlined in this review. Laboratory investigations of androgen status in men are not without pitfalls and the authors highlight problems associated with measuring and calculating serum testosterone and its fractions, the interpretation of which can be problematic. Current clinical guidelines and recommendations regarding the diagnosis and monitoring of LOH are also summarised.

Estrogen and androgen signaling in the pathogenesis of BPH.

Estrogens and androgens have both been implicated as causes of benign prostatic hyperplasia (BPH). Although epidemiological data on an association between serum androgen concentrations and BPH are inconsistent, it is generally accepted that androgens play a permissive role in BPH pathogenesis. In clinical practice, inhibitors of 5α-reductase (which converts testosterone to the more potent androgen dihydrotestosterone) have proven effective in the management of BPH, confirming an essential role for androgens in BPH pathophysiology. To date, multiple lines of evidence support a role for estrogens in BPH pathogenesis. Studies of the two estrogen receptor (ER) subtypes have shed light on their differential functions in the human prostate; ERα and ERß have proliferative and antiproliferative effects on prostate cells, respectively. Effects of estrogens on the prostate are associated with multiple mechanisms including apoptosis, aromatase expression and paracrine regulation via prostaglandin E2. Selective estrogen receptor modulators or other agents that can influence intraprostatic estrogen levels might conceivably be potential therapeutic targets for the treatment of BPH.

Testosterone testing in adult males.

The number of requests for testosterone testing in adult males has been increasing in recent years. In this review, the biochemistry and physiology of testosterone in males relevant to the chemical pathologist or clinical biochemist is outlined. The methodology for total testosterone and various laboratory tests associated with the assessment of testosterone status including free testosterone, calculated free testosterone (CFT), bioavailable testosterone (BAT) and free androgen index (FAI) is then summarised. Clinical and laboratory criteria for the diagnosis of late-onset hypogonadism (LOH) in men are critically discussed with particular emphasis on the interpretation of laboratory test results. Finally, other indications for testosterone testing in adult men such as infertility are also reviewed.

Oestrogen and benign prostatic hyperplasia: effects on stromal cell proliferation and local formation from androgen.

Oestrogens have been implicated as a cause of benign prostatic hyperplasia (BPH). Previous animal studies led to the hypothesis that oestrogens can stimulate prostate growth, resulting in hyperplasia of the gland. In humans, the precise role of oestrogens in BPH pathogenesis is currently unclear. We investigated the direct effects of oestradiol on the proliferation of BPH-derived prostate cells in culture. Oestradiol (10(-7) and 10(-6) M) moderately increased the proliferation of stromal cells in culture; this stimulation was antagonised by anti-oestrogen ICI 182 780, indicating an oestrogen receptor (ER)-mediated mechanism. By contrast, oestradiol had no effects on the proliferation of epithelial cells in culture. Parameters that can determine the response of stromal cells to oestrogens, including expression of the two ER subtypes and aromatase activity, were investigated. ER beta expression in stromal cells in culture was demonstrated by immunohistochemistry and western blot analysis, and was confirmed by semi-quantitative RT-PCR showing higher expression of ER beta than ER alpha mRNA in stromal cells. Aromatase, the enzyme that converts androgen precursors to oestrogens, was also examined. Aromatase mRNA and activity were detected in stromal, but not epithelial cells in culture, suggesting a mechanism whereby oestrogen concentrations can be regulated in the BPH stroma. Taken together, these findings support the hypothesis that oestrogens play a role in the pathogenesis of BPH, a disease characterised predominantly by stromal overgrowth.

Calculated free testosterone in men: comparison of four equations and with free androgen index.

BACKGROUND: Serum testosterone remains the most important investigation in the diagnosis of androgen deficiency in men. Most of the circulating testosterone is bound to albumin and sex hormone-binding globulin (SHBG), whereas free testosterone accounts for approximately 2% of total testosterone. Because direct measurement of free testosterone is impractical in routine practice, several equations are used to provide clinically useful estimates of free testosterone concentration. This study aimed to (1) obtain locally derived reference limits for total testosterone and calculated free testosterone (CFT) concentrations, and (2) critically evaluate the equations commonly used to estimate free testosterone. METHODS: Serum total testosterone, SHBG and albumin were assayed in morning blood samples obtained from 126 healthy men (aged 20-45 years) known to have normal semen analysis. CFT concentrations calculated using four published methods (i.e. the Sodergard, Nanjee-Wheeler, Vermeulen and Ly-Handelsman equations) were compared with one another and the free androgen index. RESULTS: Reference intervals for total testosterone and CFT by the Vermeulen equation were 9.4-31.0 nmol/L and 0.245-0.785 nmol/L (2.5-97.5 percentile), respectively. CFT values varied considerably with the four equations examined. Mean biases ranged from 5.8 to 56.0%; the Nanjee-Wheeler and Ly-Handelsman equations yielded positive and negative biases, respectively, against the other equations. Free androgen index was shown to correlate poorly with CFT (r2=0.21-0.46) and over-estimate the CFT at low SHBG concentrations. CONCLUSIONS: We have used various equations to derive reference ranges for CFT in healthy men aged 20-45 years. We suggest that CFT be incorporated into the investigation regimen for suspected hypogonadism when total testosterone results are equivocal.

Increased transcription and increased messenger ribonucleic acid (mRNA) stability contribute to increased GATA6 mRNA abundance in polycystic ovary syndrome theca cells.

CONTEXT: Polycystic ovary syndrome (PCOS) theca cells secrete increased levels of androgens. The mRNA and protein levels of the transcription factor GATA6, which regulates expression of several steroidogenic enzymes, are increased in PCOS theca cells. Thus, GATA6 is a PCOS candidate gene. OBJECTIVE: The objective of the study was to explore mechanisms by which GATA6 mRNA levels are increased in PCOS theca cells. DESIGN: Theca cell cDNA and genomic DNA from normal individuals and PCOS patients were subjected to quantitative RT-PCR and sequence analysis, respectively. SETTING: The experiments were performed in a university laboratory. PARTICIPANTS: Four hundred sixty-nine families that contain at least one PCOS patient were ascertained for genetic studies. Theca cells were obtained from four normal individuals and four PCOS patients. RESULTS: Nascent GATA6 transcript levels, which reflect GATA6 gene transcription, were significantly increased in PCOS theca cells. In normal theca cells, GATA6 mRNA has a short half-life, which was attributed to an AU-rich 3'-untranslated region sequence. The half-life of GATA6 transcripts was also significantly longer in the PCOS theca cells. However, no sequence variations in the GATA6 gene locus were associated with PCOS. CONCLUSIONS: In PCOS theca cells, GATA6 gene transcription and the stability of the GATA6 mRNA are increased. Because there is no sequence variation in the GATA6 gene locus, which is associated with PCOS, it is likely that the increased gene transcription and mRNA stability are due to intrinsic differences in PCOS theca cells.

The molecular signature of polycystic ovary syndrome (PCOS) theca cells defined by gene expression profiling.

Polycystic ovary syndrome (PCOS) is characterized by increased ovarian androgen secretion, anovulatory infertility due to arrested folliculogenesis, and is frequently found in association with insulin resistance and obesity. Characterization of PCOS theca cells demonstrated that elevated expression of the steroidogenic enzymes 17alpha hydroxylase/17,20 lyase (CYP17) and P450 side chain cleavage enzyme (CYP11A1) play a role in increased androgen production by 3beta-hydroxysteroid dehydrogenase in the PCOS theca cell. However, the gene networks and signal transduction pathways which cause the altered expansion of the steroid enzymes remain to be determined. In order to identify these gene networks and/or signaling pathways, we carried out global gene expression profiling of normal and PCOS theca cells using subtractive suppressive hybridization and oligonucleotide microarray analysis. These analyses demonstrated that approximately 2% of genes expressed in the theca cell exhibit altered mRNA abundance in PCOS. Characterization of these genes revealed that retinoic acid synthesis and Wnt signal transduction are altered in the PCOS theca cell. In addition, the transcription factor GATA6, which regulates the promoter activity of CYP17 and CYP11A, was increased in the PCOS compared to normal theca cells. Thus, global gene expression profiling has identified potential pathways which may determine the PCOS theca cell phenotype.

Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA transcription factors can lead to aberrant normalization of transfection efficiency.

BACKGROUND: Members of the GATA transcription factor family have been used in many transfection studies to investigate their roles in the regulation of gene expression. To correct for variations in transfection efficiency, the Renilla luciferase encoding plasmids pRL-TK and pRL-SV40 are commonly used as normalization controls. RESULTS: We report here that plasmids expressing GATA-4 or GATA-6 transcription factor increased Renilla luciferase gene expression by 2 to 8 fold when co-transfected with pRL-TK or pRL-SV40. This alteration of the control reporter gene activity was shown to cause erroneous normalization of transfection efficiency and thus misinterpretation of results in a transactivation assay. To circumvent the problem, we generated two mutant control plasmids from which putative GATA response elements were deleted. These deletions rendered pRL-SV40 unresponsive to GATA transcription factor stimulation and reduced the response of pRL-TK. A database search also indicates that consensus GATA binding sequences are present in other commercially available Renilla luciferase encoding plasmids; therefore, the latter can potentially be transactivated by GATA transcription factors. CONCLUSION: Taken together, these findings highlight the importance of the selection of an appropriate control reporter plasmid for the normalization of transfection efficiency.