Biological variation of immunological blood biomarkers in healthy individuals and quality goals for biomarker tests.

BACKGROUND: Cytokines, chemokines, adipocytokines, soluble cell receptors, and immune activation markers play an important role in immune responsiveness and can provide prognostic value since they reflect underlying conditions and disease states. This study was undertaken to investigate the components of biological variation for various laboratory tests of blood immunological biomarkers. RESULTS: Estimates of intra-individual coefficient of variation (CVI) and inter-individual coefficient of variation (CVG) were examined for blood immunological biomarkers. Biomarkers with CVI < 10% for both genders were CD3, CD4, and CD8 T-cells, serum levels of soluble cluster of differentiation 14 (sCD14), sCD163, and soluble glycoprotein 130 (sgp130). The CVI for serum levels of adiponectin, interleukin-1 receptor antagonist (IL-1Ra), macrophage inflammatory protein 1 beta (MIP-1ß), soluble CD40 Ligand (sCD40L), soluble interleukin-2 receptor alpha (sIL-2Rα), soluble interleukin-6 receptor (sIL-6R), soluble tumor necrosis factor receptor II (sTNF-RII), and tumor necrosis factor alpha (TNF-α) were between 11 and 20%. Biomarkers with CVG < 20% were CD3 T-cell, and serum concentrations of sCD14, sCD40L, and sgp130. The biomarkers with CVG > 40% were adiponectin, IL-1ra, leptin, MIP-1ß, sCD163, and sIL-2Rα. CONCLUSION: The biological variations of biomarkers have important monitoring value for longitudinal investigation and are essential for quality specification of tests that are performed in the laboratory. The CVI was relatively small while CVG was comparatively large and mean values of each biomarker vary between subjects. The individuality of biomarkers significantly influences reference interval values. A majority of the biomarkers in this study had strong individuality and the result of each biomarker should be cautiously interpreted if using established reference interval values. Comparison of a patient's test result with previous ones may be more useful than the usage of conventional reference values.

Stability of cytokines, chemokines and soluble activation markers in unprocessed blood stored under different conditions.

BACKGROUND: Biomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24h at room and refrigerator temperature. METHODS: Blood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20-25°C) and refrigerator temperature (4-8°C) for 0.5, 2, 4, 6, 8, and 24h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation. RESULTS: IFN-γ, sIL-2Rα, sTNF-RII and ß2-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24h. IL-6, TNF-α, MIP-1ß and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1ß were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature. CONCLUSION: All the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing.

Defining the role of albumin infusion in cirrhosis-associated hyponatremia.

The presence of negatively charged, impermeant proteins in the plasma space alters the distribution of diffusible ions in the plasma and interstitial fluid (ISF) compartments to preserve electroneutrality and is known as Gibbs-Donnan equilibrium. In patients with hypoalbuminemia due to underlying cirrhosis, the decrease in the plasma water albumin concentration ([Alb-]pw) would be expected to result in a decrease in the plasma water sodium concentration ([Na+]pw) due to an alteration in the distribution of Na+ between the plasma and ISF. In addition, cirrhosis-associated hyponatremia may be due to the renal diluting defect resulting from the intravascular volume depletion due to gastrointestinal losses and overdiuresis and/or decreased effective circulatory volume secondary to splanchnic vasodilatation. Therefore, albumin infusion may result in correction of the hyponatremia in cirrhotic patients either by modulating the Gibbs-Donnan effect due to hypoalbuminemia or by restoring intravascular volume in patients with intravascular volume depletion due to gastrointestinal losses and overdiuresis. However, the differential role of albumin infusion in modulating the [Na+]pw in these patients has not previously been analyzed quantitatively. In the present study, we developed an in vitro assay system to examine for the first time the quantitative effect of changes in albumin concentration on the distribution of Na+ between two compartments separated by a membrane that allows the free diffusion of Na+. Our findings demonstrated that changes in [Alb-]pw are linearly related to changes in [Na+]pw as predicted by Gibbs-Donnan equilibrium. However, based on our findings, we predict that the improvement in cirrhosis-associated hyponatremia due to intravascular volume depletion results predominantly from the restoration of intravascular volume rather than alterations in Gibbs-Donnan equilibrium.

Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free ß-subunit, and ß-subunit core fragment in urine.

BACKGROUND: Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free ß-subunit (hCGß), and ß-subunit core fragment (hCGßcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential immunoextraction method with LC-MS/MS detection for quantification of intact hCG, hCGß, and hCGßcf in urine. METHODS: hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCGß and hCGßcf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. RESULTS: The method was linear to 20 IU/L. Total imprecision was 6.6%-13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4-15.5 IU/L higher than LC-MS/MS results. CONCLUSIONS: The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCGß separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

Interlaboratory agreement of insulin-like growth factor 1 concentrations measured by mass spectrometry.

BACKGROUND: Insulin-like growth factor 1 (IGF-1)(7) is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported. METHODS: We deployed an LC-MS/MS method to quantify serum IGF-1 in 5 laboratories using 5 different instruments and analyzed 130 healthy human samples and 22 samples from patients with acromegaly. We determined measurement imprecision (CV) for differences due to instrumentation, calibration curve construction, method of calibration, and reference material. RESULTS: Instrument-dependent variation, exclusive of digestion, across 5 different instrument platforms was determined to be 5.6%. Interlaboratory variation was strongly dependent on calibration. Calibration materials from a single laboratory resulted in less variation than materials made in individual laboratories (CV 5.2% vs 12.8%, respectively). The mean imprecision for 152 samples between the 5 laboratories was 16.0% when a calibration curve was made in each laboratory and 11.1% when a single-point calibration approach was used. CONCLUSIONS: The interlaboratory imprecision of serum IGF-1 concentrations is acceptable for use of the assay in antidoping laboratories and in standardizing results across clinical laboratories. The primary source of variability is not derived from the sample preparation but from the method of calibration.

Serum albumin and kidney function decline in HIV-infected women.

BACKGROUND: Serum albumin concentrations are a strong predictor of mortality and cardiovascular disease in human immunodeficiency virus (HIV)-infected individuals. We studied the longitudinal associations between serum albumin levels and kidney function decline in a population of HIV-infected women. STUDY DESIGN: Retrospective cohort analysis. SETTING & PARTICIPANTS: Study participants were recruited from the Women's Interagency HIV Study (WIHS), a large observational study designed to understand risk factors for the progression of HIV infection in women living in urban communities. 908 participants had baseline assessment of kidney function and 2 follow-up measurements over an average of 8 years. PREDICTOR: The primary predictor was serum albumin concentration. OUTCOMES: We examined annual change in kidney function. Secondary outcomes included rapid kidney function decline and incident reduced estimated glomerular filtration rate (eGFR). MEASUREMENTS: Kidney function decline was determined by cystatin C-based (eGFR(cys)) and creatinine-based eGFR (eGFR(cr)) at baseline and follow-up. Each model was adjusted for kidney disease and HIV-related risk factors using linear and relative risk regression. RESULTS: After multivariate adjustment, each 0.5-g/dL decrement in baseline serum albumin concentration was associated with a 0.56-mL/min faster annual decline in eGFR(cys) (P < 0.001), which was attenuated only slightly to 0.55 mL/min/1.73 m(2) after adjustment for albuminuria. Results were similar whether using eGFR(cys) or eGFR(cr). In adjusted analyses, each 0.5-g/dL lower baseline serum albumin level was associated with a 1.71-fold greater risk of rapid kidney function decline (P < 0.001) and a 1.72-fold greater risk of incident reduced eGFR (P < 0.001). LIMITATIONS: The cohort is composed of only female participants from urban communities within the United States. CONCLUSIONS: Lower serum albumin levels were associated strongly with kidney function decline and incident reduced eGFRs in HIV-infected women independent of HIV disease status, body mass index, and albuminuria.

Does the analysis of the enantiomeric composition of clenbuterol in human urine enable the differentiation of illicit clenbuterol administration from food contamination in sports drug testing?

RATIONALE: Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzyl alcohol) is approved for human and veterinary use primarily for the treatment of pulmonary afflictions. Despite the authorized administration in cases of medical indications, the misuse of clenbuterol in animal husbandry as well as elite and amateur sport has frequently been reported, arguably due to growth-promoting properties. Due to various recent incidences of doping control specimens containing clenbuterol, strategies towards the discrimination of a surreptitious application from unintended intake via animal-derived edibles or dietary supplements were required. METHODS: The enantiomeric compositions of clenbuterol in human urine samples derived from administration studies with therapeutic amounts of the ß(2)-agonist and authentic doping control specimens were determined. Due to the facts that therapeutic clenbuterol consists of a racemic mixture of (+)- and (-)-stereoisomers and that the first mentioned (dextrorotatory) stereoisomer is retained to a greater extent in edible animal tissue, the differentiation of a recent administration of therapeutic (and thus racemic) clenbuterol from food contamination (stereoisomerically depleted clenbuterol) was considered. Employing deuterated clenbuterol as internal standard, the target analytes were extracted from human urine by means of concerted liquid-liquid and solid-phase extractions and subjected to chiral liquid chromatography hyphenated to high resolution/high accuracy mass spectrometry with electrospray ionization. RESULTS: Both enantiomers of clenbuterol were baseline separated and relative abundances of corresponding labeled and unlabeled stereoisomers were determined, demonstrating that the therapeutic use of clenbuterol results in racemic mixtures in urine for at least 24 h while adverse analytical findings presumably originating from food contaminations can yield (-)-clenbuterol-depleted pairs of analytes. CONCLUSIONS: The determination of relative abundances of clenbuterol enantiomers can indicate the ingestion of clenbuterol via contaminated food; however, depletion of (-)-clenbuterol in edible animal tissue is time-dependent and thus results can still be inconclusive as to the inadvertent ingestion of clenbuterol when clenbuterol administration to animals was conducted until slaughter.

Sex hormone-binding globulin and risk of clinical diabetes in American black, Hispanic, and Asian/Pacific Islander postmenopausal women.

BACKGROUND: Recent prospective studies have shown a strong inverse association between sex hormone-binding globulin (SHBG) concentrations and risk of clinical diabetes in white individuals. However, it remains unclear whether this relationship extends to other racial/ethnic populations. METHODS: We evaluated the association between baseline concentrations of SHBG and clinical diabetes risk in the Women's Health Initiative Observational Study. Over a median follow-up of 5.9 years, we identified 642 postmenopausal women who developed clinical diabetes (380 blacks, 157 Hispanics, 105 Asians) and 1286 matched controls (777 blacks, 307 Hispanics, 202 Asians). RESULTS: Higher concentrations of SHBG at baseline were associated with a significantly lower risk of clinical diabetes [relative risk (RR), 0.15; 95% CI, 0.09-0.26 for highest vs lowest quartile of SHBG, adjusted for BMI and known diabetes risk factors]. The associations remained consistent within ethnic groups [RR, 0.19 (95% CI, 0.10-0.38) for blacks; RR, 0.17 (95% CI, 0.05-0.57) for Hispanics; and 0.13 (95% CI, 0.03-0.48) for Asians]. Adjustment for potential confounders, such as total testosterone (RR, 0.11; 95% CI, 0.07-0.19) or HOMA-IR (RR, 0.26; 95% CI, 0.14-0.48) did not alter the RR substantially. In addition, SHBG concentrations were significantly associated with risk of clinical diabetes across categories of hormone therapy use (never users: RR(per SD) = 0.42, 95% CI, 0.34-0.51; past users: RR(per SD) = 0.53;, 95% CI, 0.37-0.77; current users: RR(per SD) = 0.57; 95% CI, 0.46-0.69; P-interaction = 0.10). CONCLUSIONS: In this prospective study of postmenopausal women, we observed a robust, inverse relationship between serum concentrations of SHBG and risk of clinical diabetes in American blacks, Hispanics, and Asians/Pacific Islanders. These associations appeared to be independent of sex hormone concentrations, adiposity, or insulin resistance.

Rapid flow cytometry-based structural maintenance of chromosomes 1 (SMC1) phosphorylation assay for identification of ataxia-telangiectasia homozygotes and heterozygotes.

BACKGROUND: No rapid reliable method exists for identifying ataxia-telangiectasia (A-T) homozygotes or heterozygotes. Heterozygotes are at an increased risk of cancer and are more sensitive to the effects of ionizing radiation (IR) than the general population. We report a rapid flow cytometry (FC)-based ataxia-telangiectasia mutated (ATM) kinase assay that measures ATM- dependent phosphorylation of structural maintenance of chromosomes 1 (SMC1) following DNA damage (FC-pSMC1 assay). METHODS: After optimizing conditions with lymphoblastoid cell lines (LCLs), we studied peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy donors (unknowns), 10 obligate A-T heterozygotes, and 6 unrelated A-T patients. One hour after DNA damage (by either IR or bleomycin), the cells were fixed and incubated with a primary antibody to SMC1pSer966. We analyzed the stained cells by FC to determine the difference in geometric mean fluorescence intensity (DeltaGMFI) of untreated and treated cells; this difference was expressed as a percentage of daily experimental controls. RESULTS: The FC-pSMC1 assay reliably distinguished ATM heterozygotes and homozygotes from controls. Average DeltaGMFI percentages (SD) of daily controls were, for unknowns, 106.1 (37.6); for A-T heterozygotes, 37.0 (18.7); and for A-T homozygotes; -8.73 (16.2). Values for heterozygotes and homozygotes were significantly different from those of controls (P < 0.0001). CONCLUSIONS: The FC-pSMC1 assay shortens the turnaround time for diagnosing A-T homozygotes from approximately 3 months to approximately 3 h. It also identifies A-T heterozygotes and can be used for prenatal counseling or for screening individuals in large study cohorts for potential ATM heterozygosity, which can then be confirmed by sequencing.

Methemoglobin effects on coagulation: a dose-response study with HBOC-200 (Oxyglobin) in a thrombelastogram model.

OBJECTIVES: Because oxidation affects platelet and coagulation factors, hemoglobin auto-oxidation in HBOCs results in the transformation to methemoglobin, which may have additive adverse effects on coagulation. The risk of coagulopathy after different dilutions of HBOC-200 with low and high methemoglobin concentrations was studied. DESIGN: A laboratory study on donor blood using thromboelastography (TEG; Haemoscope, Niles, IL). SETTING: A university laboratory. PARTICIPANTS: Volunteer donor blood. INTERVENTIONS: Blood samples simulated hemodilution during clinical resuscitation of hemorrhagic shock with varying doses of HBOC-200 (Oxyglobin; Biopure Corp, Cambridge, MA). Coagulopathy related to 1:11, 1:5, 1:2, and 1:1 dilution of whole blood with HBOC-200 high methemoglobin concentrations (65%) and HBOC-200 low methemoglobin concentrations (1%) were analyzed. MEASUREMENTS AND MAIN RESULTS: Analysis of fixed effects of dilution on coagulation showed that the progressive dilution of HBOC-200 (low methemoglobin) and HBOC-200 (high methemoglobin) produced significant prolongation in reaction time (R) and clot propagation (K) and significant decreases in clot kinetics (alpha) and clot strength (MA and G). Analysis of fixed effects of treatment group on coagulation showed that clot propagation (K, alpha) and clot strength (MA and G) are significantly different in HBOC-200 (high methemoglobin) compared with HBOC-200 (low methemoglobin). CONCLUSIONS: High methemoglobin concentrations in HBOC-200 cause additive coagulation impairment that likely results from the effects of oxidative substances on platelet function and coagulation proteins. Oxidative products adversely react with coagulation factors and modify redox-sensitive sites in the platelets. Therefore, if methemoglobinemia occurs as a result of HBOC administration and if the levels are significantly elevated (greater than 10%), impairment of coagulation is possible.

The influence of EDTA Vacutainer blood collection tube on the level of blood interleukin-1 receptor antagonist.

BACKGROUND: The use of anticoagulants may influence the composition of blood cells and interfere with plasma levels of IL-1ra when unprocessed EDTA blood samples are stored for long periods of time. METHODS: Blood was drawn into EDTA and heparinized blood collection tubes from 11 HIV-1 negative men participating in the Multicenter AIDS Cohort Study (MACS) and 4 healthy volunteers. The blood was processed according to the experiments detailed in the method and after incubation; supernatants were collected and stored at -70 °C until batch testing using IL-1ra ELISA. RESULTS: There was no difference between the levels of IL-1ra in EDTA blood collected into plastic and glass tubes (p = .911). There were significant increases from baseline levels of IL-1ra (p ≤ .05) after 24 h incubation for diluted whole blood and PBMC supernatants but not for granulocytes supernatants. CONCLUSION: EDTA as an anticoagulant influences the blood concentrations of IL-1ra in unprocessed blood. Thus, EDTA blood is not a suitable specimen for measurement of IL-1ra. Other types of anticoagulated blood should be processed within one hour of draw whenever measuring plasma levels of IL-1ra.

A multicenter evaluation of the Iris iQ200 automated urine microscopy analyzer body fluids module and comparison with hemacytometer cell counts.

Manual hemacytometer cell counting of body fluids is labor-intensive and requires skilled testing personnel. We performed a multicenter evaluation of the Iris iQ200 automated microscopy analyzer Body Fluids Module (Iris Diagnostics, Chatsworth, CA) and compared 350 iQ200 body fluid cell counts with manual hemacytometer cell counts. Within-run imprecision, expressed as coefficient of variation (CV), ranged from 2.6% to 5.9% for RBC counts between 875 and 475 x 10(6)/L and from 4.2% to 6.5% for nucleated cell counts between 820 and 590 x 10(6)/L. The lower limits of detection, based on a CV of 20% or less, were 30 and 35 x 10(6)/L for RBCs and nucleated cells, respectively. There was very good agreement between automated iQ200 and manual body fluid cell counts based on slopes and r2 values. The iQ200 has satisfactory performance for enumerating RBCs and nucleated cells in most body fluids, with the exception of cerebrospinal fluid specimens that contain low cell numbers.

Does Hextend impair coagulation compared to 6% hetastarch? An ex vivo thromboelastography study.

The objective of this study was to determine if coagulation is different between 6% hetastarch in normal saline (NS) and 6% hetastarch in lactated Ringer's solution (LR), with use of an ex vivo thromboelastography (TEG) model with healthy donated volunteer blood. We simulated hemodilution that occurs during clinical resuscitation of hemorrhagic or hypovolemic shock, using healthy human donor whole blood (WB) ex vivo. Coagulopathy related to low, medium, high, or very high dilution of WB with NS or a high-molecular-weight hetastarch-based plasma expander, 6% hetastarch in NS (HSNS) or 6% hetastarch in lactated Ringer's [Hextend (HSLR)], was analyzed by thromboelastography (TEG). No changes were noted in the TEG profile of undiluted WB controls during the 6-hour period of use (P > 0.95). Dilution with HSNS and HSLR significantly impaired coagulation compared to both WB control and NS. Progressive dilution with NS impaired coagulation but to a lesser extent than colloids (P < 0.01). Low dilution of blood with NS increased clot strength by 12% (not significant; P = 0.097). We conclude that WB containing citrate obtained from healthy donors for TEG analysis yields reproducible data over a minimum of 6 hours. Either hetastarch, when present at concentrations comparable to the manufacturer's maximum recommended dose of 20 mL/kg (equivalent to the high dilution used in these experiments), decreases clot tensile strength to levels associated with an increased risk of bleeding. Substitution of lactated Ringer's for NS in 6% hetastarch appears to offer no advantage in avoiding hemostatic compromise in an in vitro model.

Does OxyVita, a new-generation hemoglobin-based oxygen carrier, or oxyglobin acutely interfere with coagulation compared with normal saline or 6% hetastarch? An ex vivo thromboelastography study.

OBJECTIVES: Because hetastarches have deleterious effects on coagulation that increase with molecular weight (MWt), risk of coagulopathy associated with a high MWt hemoglobin-based oxygen carrier (HBOC) was studied. DESIGN: Preliminary laboratory study of donor blood using thromboelastography (TEG). SETTING: University laboratory. PARTICIPANTS: Volunteer donor blood. INTERVENTIONS: Experiments simulated hemodilution during clinical resuscitation of hemorrhagic shock with varying doses of HBOCs. Coagulopathy related to 1:11, 1:5, 1:2, or 1:1 dilution of whole blood with normal saline, 6% hetastarch (670 kilodaltons [kD]), hemoglobin glutamer-200 (HBOC-200, 200 kD), or OxyVita (OXYVITA Inc, New Windsor, NY) (a new-generation, zero-link polymerized bovine hemoglobin-based oxygen carrier, 33 megadaltons) were analyzed. MEASUREMENTS AND MAIN RESULTS: At 2 lower levels of hemodilution, hetastarch, HBOC-200, and OxyVita produced equivalent reductions in maximum clot strength (TEG-MA and TEG-G) that reached statistical significance compared with whole blood and normal saline. At 2 higher dilutions, OxyVita and HBOC-200 impaired maximum clot strength compared with whole blood, normal saline, and hetastarch. Dilution with hetastarch had a greater effect on clot propagation (K and alpha) than either HBOC. CONCLUSIONS: OxyVita and HBOC-200, HBOCs with different MWt, had similar effects on coagulation as measured by TEG. The impairment of coagulation by HBOCs and hetastarch occurred at doses corresponding to 12 mL/kg or a blood volume replacement of 17%. The use of HBOCs at doses corresponding to 23 mL/kg or a blood volume replacement of 33% significantly decreased coagulation to levels associated with increased clinical bleeding in this preliminary study. Minimal coagulopathic effects are expected with use of OxyVita at the manufacturer's anticipated effective dose of 10 g or 2 to 3 mL/kg.

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction-tandem mass spectrometry to detect hCG use.

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti-Doping Agency (WADA) list of prohibited substances. The majority of WADA-accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study we measured the concentration of intact hCG, free ß-subunit (hCGß) and ß-subunit core fragment (hCGßcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC-MS/MS method. Mean concentrations of intact hCG, hCGß and hCGßcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGß and hCGßcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.

The influence of small doses of ethanol on the urinary testosterone to epitestosterone ratio in men and women.

Endogenous steroid use can increase urinary testosterone/epitestosterone (T/E) values. In addition, ethanol in amounts >0.5 g per kg of body weight (g/kg) can also increase T/E values. However, the effect of smaller doses of ethanol on T/E values is unknown. The influence of 0.2 and 0.4 g/kg of ethanol on baseline T/E values in 20 men and 20 women with low and high baseline T/E values was investigated and correlated with ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations. T/E values for 7 of the women were excluded from the study because of undetectable T concentrations or for other reasons. One man and 1 woman with a high T/E baseline value had a significant increase in their T/E value after ingestion of 0.2 g/kg of ethanol. One man and 2 women with a high T/E baseline, and 1 woman with a low T/E baseline had significantly increased T/E values after ingestion of 0.4 g/kg of ethanol. There was wide variability in peak EtG concentrations and a lack of correlation between ethanol dose and EtG concentrations. Interestingly, 1 man and 2 women with increased T/E values following ethanol ingestion had EtG concentrations below the World Anti-Doping Agency (WADA) cut-off of 5000 ng/mL. These findings demonstrate that small amounts of ethanol can elevate T/E values, with women being more susceptible. In addition, consideration should be given to the lowering of the WADA EtG cut-off to detect samples with elevated T/E values from ingestion of low doses of ethanol.

Relationships of sex hormone levels with leukocyte telomere length in Black, Hispanic, and Asian/Pacific Islander postmenopausal women.

BACKGROUND: Sex hormones may play important roles in sex-specific biological aging. In the study, we specifically examined associations between circulating sex hormone concentrations and leukocyte telomere length (TL). METHODS: A cross-sectional study was conducted among 1124 Black, 444 Hispanic, and 289 Asian/Pacific Islander women in the Women's Health Initiative Observational Cohort. Estradiol and testosterone concentrations were measured using electrochemiluminescence immunoassays; TL was measured using quantitative polymerase chain reaction. RESULTS: Women in the study were aged 50-79 years. Estradiol concentrations were not significantly associated with TL in this sample. The associations between total and free testosterone and TL differed by race/ethnicity (Pinteraction = 0.03 and 0.05 for total and free testosterone, respectively). Total and free testosterone concentrations were not associated with TL in Black and Hispanic women, whereas in Asian/Pacific Islander women their concentrations were inversely associated with TL (Ptrend = 0.003 for both). These associations appeared robust in multiple subgroup analyses and multivariable models adjusted for potential confounding factors. In Asian/Pacific Islander women, a doubling of serum free and total testosterone concentrations was associated with a 202-bp shorter TL (95% confidence interval [CI] 51-353 bp) and 203-bp shorter TL (95% CI 50-355 bp), respectively. CONCLUSIONS: Serum estradiol concentrations were not associated with leukocyte TL in this large sample of postmenopausal women. Total and free testosterone concentrations were inversely associated with TL in Asian/Pacific Islander women, but not in Black and Hispanic women, although future studies to replicate our observations are warranted particularly to address potential ethnicity-specific relationships.

Falsely elevated thyroid hormone levels due to anti-sheep antibody interference in an automated electrochemiluminescent immunoassay.

We describe a 43-year-old woman with falsely increased thyroxine (T(4)) and triiodothyronine (T(3)) concentrations (total and index values) using a competitive electrochemiluminescent immunoassay, due to human anti-sheep antibodies. The thyrotropin (TSH) concentration was within normal limits. When specimens were re-tested by an immunoassay utilizing mouse antibodies, the total T(4) and T(3) concentrations were within normal limits. Removal of IgG by protein G column chromatography resulted in normalization of total T(4) and T(3) concentrations. In contrast, a mouse IgG column failed to normalize the elevated total T(4) and T(3) concentrations. Other immunoassays utilizing mouse monoclonal antibodies and rabbit antisera were unaffected, indicating that the interference was anti-sheep antibodies and not heterophile antibodies. We believe this is the first report of human anti-sheep antibodies causing falsely increased total and free T(4) and T(3) serum concentrations in competitive immunoassays using sheep antisera. Clinicians need to be aware of this potential problem since inaccurate thyroid function tests can lead to inappropriate treatment decisions.

Association of Kidney Function and Early Kidney Injury With Incident Hypertension in HIV-Infected Women.

Subclinical kidney disease is associated with developing hypertension in the general population, but data are lacking among HIV-infected people. We examined associations of kidney function and injury with incident hypertension in 823 HIV-infected and 267 HIV-uninfected women in the Women's Interagency HIV Study, a multicenter, prospective cohort of HIV-infected and uninfected women in the United States. Baseline kidney biomarkers included estimated glomerular filtration rate using cystatin C, urine albumin-to-creatinine ratio, and 7 urine biomarkers of tubular injury: α-1-microglobulin, interleukin-18, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, liver fatty acid-binding protein, N-acetyl-ß-d-glucosaminidase, and α1-acid-glycoprotein. We used multivariable Poisson regression to evaluate associations of kidney biomarkers with incident hypertension, defined as 2 consecutive visits of antihypertensive medication use. During a median follow-up of 9.6 years, 288 HIV-infected women (35%) developed hypertension. Among the HIV-infected women, higher urine albumin-to-creatinine ratio was independently associated with incident hypertension (relative risk =1.13 per urine albumin-to-creatinine ratio doubling, 95% confidence interval, 1.07-1.20), as was lower estimated glomerular filtration rate (relative risk =1.10 per 10 mL/min/1.73 m2 lower estimated glomerular filtration rate; 95% confidence interval, 1.04-1.17). No tubular injury and dysfunction biomarkers were independently associated with incident hypertension in HIV-infected women. In contrast, among the HIV-uninfected women, urine albumin-to-creatinine ratio was not associated with incident hypertension, whereas higher urine interleukin-18, α1-acid-glycoprotein, and N-acetyl-ß-d-glucosaminidase levels were significantly associated with incident hypertension. These findings suggest that early glomerular injury and kidney dysfunction may be involved in the pathogenesis of hypertension in HIV-infected people. The associations of tubular markers with hypertension in HIV-uninfected women should be validated in other studies.