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.

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.

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.

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.

Macrophage Inflammatory Protein-3 Alpha (MIP-3α)/CCL20 in HIV-1-Infected Individuals.

OBJECTIVE: Uncontrolled HIV infection progresses to the depletion of systemic and mucosal CD4 and AIDS. Early HIV infection may be associated with increases in the concentration of MIP-3α in the blood and gut fluids. MIP-3α/CCL20 is the only chemokine known to interact with CCR6 receptors which are expressed on immature dendritic cells and both effector and memory CD8+ and CD4+ T cells. The role and prognostic value of blood levels of MIP-3α in HIV-infected individuals has yet to be described. METHODS: We determined the serum levels of MIP-3α, and IFN-γ, in 167 HIV-1-infected and 27 HIV-1-uninfected men participating in the Multicenter AIDS Cohort Study (MACS). The blood biomarkers were measured using enzyme-linked immunosorbent assays (ELISA) and the cell phenotypes using flow cytometry. RESULTS: Median serum levels of MIP-3α in HIV-1-infected and uninfected men was significantly different (p<0.0001) and were 21.3 pg/mL and 6.4 pg/mL respectively. The HIV-1-infected men with CD4+ T cell count <200 cells/µL showed the highest median serum MIP-3α (23.1 pg/mL). Serum levels of MIP-3α in HIV-1 infected (n=167) were negatively correlated with absolute number of CD4+ T cell (p=0.01) and were positively correlated with CD38 molecules on CD8+ T cells (p=0.0002) and with serum levels of IFN-γ (0.006). CONCLUSION: Serum levels of MIP-3α concomitantly increase with plasma levels of IFN-γ, CD38 expression on CD8+ T cells, and decreased of absolute CD4+ T cells in HIV-1-infected men. A higher blood level of MIP-3α may be representation of locally high level of MIP-3α and more recruitment of immature dendritic cell at site of infection. Involvement of CCR6/CCL20 axis and epithelial cells at the recto-colonel level may enhance sexual transmission of HIV-1 in MSM and may be useful as a prognostic marker in HIV-1-infection and AIDS.

Urinary human chorionic gonadotropin isoform concentrations in doping control samples.

Anti-doping laboratories routinely use immunoassays to measure urinary concentrations of human chorionic gonadotropin (hCG). To minimize immunoassay differences and false positive screen results from inactive isoforms (free ß-subunit (hCGß), ß-subunit core fragment (hCGßcf)) laboratories now use intact hCG instead of total hCG immunoassays to measure hCG. To determine the distribution of hCG isoforms in urine, we determined the concentrations of intact hCG, hCGß, and hCGßcf in male urine samples based on immunoassay total hCG concentrations using a sequential immunoextraction and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. hCG was isolated using antibody-conjugated magnetic beads and unique tryptic peptides were quantified by LC-MS/MS. Negative samples with detectable but low total hCG concentrations (1.2-3.5 pmol/L) had intact and hCGß concentrations <1.2 pmol/L, and hCGßcf concentrations <2.3 pmol/L by LC-MS/MS. Urine samples from an athlete receiving hCG had intact hCG concentrations ranging from 18.8 to 57.6 pmol/L, hCGß concentrations <0.7 pmol/L, and hCGßcf concentrations ranging from 94 to 243% of the intact hCG concentration. In 27 atypical samples with total hCG concentrations ranging from 16.7 to 412.7 pmol/L with intact hCG <2.7 pmol/L by immunoassay, all samples had intact hCG concentrations <3.8 pmol/L and hCGß concentrations <6.2 pmol/L by LC-MS/MS. hCGßcf concentrations by LC-MS/MS varied widely and ranged from 1.03 to 21.9 pmol/L. In summary, total hCG immunoassays significantly overestimate hCG concentrations and can produce false positive results. Although the intact hCG immunoassay slightly overestimates hCG concentrations compared to LC-MS/MS, it can distinguish between cases of hCG use and atypical cases with elevated total hCG concentrations. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of 2 Batched Pretreatment Systems for the Measurement of Whole Blood Tacrolimus on the ARCHITECT Immunoassay Analyzer.

BACKGROUND: Measurement of tacrolimus using the ARCHITECT immunoassay analyzer requires a manual extraction step that puts clinical laboratory workers at risk for ergonomic injury. Therefore, we developed 2 batched extraction systems for tacrolimus measurement on the ARCHITECT analyzer and describe their features herein. METHODS: Two batched extraction methods were developed at 2 different laboratories. The batched extraction methods allow processing of at least 20 specimens at a time. We evaluated the analytical performance of those methods and compared them with the United States Food and Drug Administration (FDA)-cleared process for manually extracting individual specimens. RESULTS: Comparing the performance of batched- and individual-extraction methods revealed that both methods had comparable between-day imprecision, high patient-results correlation (R2 values ≥0.9869), equivalent functional sensitivity (0.48 ng/mL), and good linearity between 1 ng per mL and 25 ng per mL. Further, we observed decreased delta check-identified errors using the batched method. CONCLUSION: The 2 developed batched extraction methods for tacrolimus measurement that we describe herein demonstrate excellent performance and can replace individual specimen extraction.

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.

The Urine Marker Test: An Alternative Approach to Supervised Urine Collection for Doping Control.

BACKGROUND: Urine sample collection for doping control tests is a key component of the World Anti-Doping Agency's fight against doping in sport. However, a substantial number of athletes experience difficulty when having to urinate under supervision. Furthermore, it cannot always be ensured that athletes are actually delivering their own urine. A method that can be used to alleviate the negative impact of a supervised urination procedure and which can also identify urine as coming from a specific athlete is the urine marker test. Monodisperse low molecular weight polyethylene glycols (PEGs) are given orally prior to urination. Urine samples can be traced to the donor by analysis of the PEGs previously given. OBJECTIVE: The objective of this study was to investigate the use of the urine marker during urine doping control testing. METHODS: Two studies investigated athletes' acceptance of this new method via two questionnaires (n = 253). Furthermore, a third study (n = 91) investigated whether ingestion of the marker can identify the urine as coming from a specific person and whether the marker interferes with the detection of prohibited substances. RESULTS AND CONCLUSIONS: The results indicate that this new method finds wide acceptance both from athletes who have only heard about the procedure and those who have actually tested the new method. Furthermore, the marker, which can identify urine as coming from a specific person, does not interfere with the detection of prohibited substances.

Estimating the hCGßcf in urine during pregnancy.

OBJECTIVE: Elevated urine concentrations of hCG beta core fragment (hCGßcf) are known to cause false negative qualitative point-of-care hCG test results, but limited information is available regarding urine hCGßcf. In this study, we evaluate the relationship between serum and urine hCG concentrations and the frequency of elevated urine hCGßcf concentrations. DESIGN AND METHODS: Paired serum and urine specimens were obtained from 60 women at various stages of pregnancy and hCG was measured using the Abbott Architect and Roche Cobas e602 assays. Urine specimens with the greatest difference in urine hCG concentrations between these two instruments were tested using a qualitative point-of-care device and hCGßcf was quantified using LC-MS/MS. RESULTS: Urine hCG concentrations were lower than serum and the magnitude of the difference depended on whether the hCG assay detected hCGßcf. Elevated hCGßcf concentrations (>280,000pmol/L) were observed in 12% of specimens from an unselected patient population. There was a significant correlation (r=0.97; p<0.0001) between the difference (Roche hCG-Abbott hCG) and the hCGßcf concentration as measured by LC-MS/MS (Roche-Abbott difference IU/L=(hCGßcf (pmol/L)∗0.131+656)). CONCLUSIONS: A correlation exists between serum and urine hCG concentrations but this correlation is variable. hCGßcf concentrations can be estimated using two automated assay reagent platforms that differ in their recognition of hCGßcf.

Detection of Stimulants and Narcotics by Liquid Chromatography-Tandem Mass Spectrometry and Gas Chromatography-Mass Spectrometry for Sports Doping Control.

Sports drug testing laboratories are required to detect several classes of compounds that are prohibited at all times, which include anabolic agents, peptide hormones, growth factors, beta-2 agonists, hormones and metabolic modulators, and diuretics/masking agents. Other classes of compounds such as stimulants, narcotics, cannabinoids, and glucocorticoids are also prohibited, but only when an athlete is in competition. A single class of compounds can contain a large number of prohibited substances and all of the compounds should be detected by the testing procedure. Since there are almost 70 stimulants on the prohibited list it can be a challenge to develop a single screening method that will optimally detect all the compounds. We describe a combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) testing method for detection of all the stimulants and narcotics on the World Anti-Doping Agency prohibited list. Urine for LC-MS/MS testing does not require sample pretreatment and is a direct dilute and shoot method. Urine samples for the GC-MS method require a liquid-liquid extraction followed by derivatization with trifluoroacetic anhydride.

Evaluation of a semi-quantitative pregnancy device for susceptibility to interference caused by hCGßcf.

OBJECTIVE: Previous work has documented the ability of the Clearblue Advanced Test with Weeks Estimator, a new over-the-counter (OTC) urine hCG device, to accurately estimate weeks since ovulation in early pregnancy. In this study, the performance of this device in more advanced pregnancy was assessed. METHODS: The Clearblue Advanced Test with Weeks Estimator device was used to test solutions containing purified intact hCG and hCGßcf at concentrations consistent with early, middle and late pregnancy. Urine samples from three normal pregnant patients 9-13 weeks of gestation and from a patient 12 weeks of gestation known to generate false negative results on qualitative urine test devices due to excess hCGßcf were also evaluated. RESULTS: The Clearblue Weeks Estimator device gave expected results using solutions containing purified intact hCG and hCGßcf at concentrations observed throughout pregnancy. The device generated expected results using urine from three of four patients tested between 9 and 13 weeks of gestation. However, when urine from a patient with elevated concentrations of hCGßcf was used, the device correctly indicated pregnancy although the estimate for the date was incorrect. CONCLUSION: This device gave expected "pregnant" results using all samples tested. However, the "Weeks Estimator" should be interpreted with caution when used by patients after seven weeks of pregnancy.

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.

Monoclonal and polyclonal immunoglobulin interference in a conjugated bilirubin assay.

CONTEXT: Monoclonal proteins can interfere with the conjugated bilirubin assay on the Beckman Coulter AU5400 and AU2700 instruments and produce spurious results. Protein depletion eliminates the interference, suggesting that monoclonal proteins cause the problem. OBJECTIVES: To determine the interference rate with the Beckman Coulter AU5400 and AU2700 conjugated bilirubin assay and to identify the interfering substance. DESIGN: Beckman Coulter bilirubin results from 33,720 samples analyzed during 6 months were evaluated for interference. On a subset of samples, protein G columns were used to specifically remove immunoglobulin G (IgG) to determine the cause of the interference. Another 117 samples containing a (known) monoclonal protein were selected to determine the interference rate. RESULTS: From 26 different patients, 103 samples had conjugated bilirubin results greater than the total bilirubin for a false-positive rate of 0.3%. Removal of IgG from a subset of those samples with IgG monoclonal protein and increased polyclonal IgG eliminated the conjugated bilirubin interference. In separate, selected samples containing monoclonal proteins, the false-positive interference rate was 7.7% (9 of 117) and was greatest when the monoclonal protein concentration was greater than 4 g/dL. Another 11 of the 117 samples (9.4%) with monoclonal proteins exhibited assay interference by producing false-negative results. The overall interference rate, therefore, was 17.1%. CONCLUSION: The false-positive interference rate for the Beckman Coulter conjugated bilirubin assay was 0.3% for routinely analyzed serum/plasma samples. In addition to monoclonal proteins, we found that polyclonal immunoglobulins can also interfere with the conjugated bilirubin assay. The overall interference rate was 17.1% for samples with a monoclonal protein.

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.

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.

Association of Blood Biomarkers of Bone Turnover in HIV-1 Infected Individuals Receiving Anti-Retroviral Therapy (ART).

OBJECTIVE: To evaluate the association of bone turnover biomarkers with blood levels of alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BAP), osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP), parathyroid hormone (PTH), and other blood markers in HIV-1 infected men receiving anti-retroviral therapy (ART). Advances in the treatment of HIV-1 infection have extended the life span of HIV-1 infected individuals. However, these advances may come at the price of metabolic side effects and bone disorders, including premature osteopenia, osteoporosis and osteonecrosis. METHODS: Analyses of Ostase BAP, osteocalcin, and TRAP in blood were measured in three groups of MACS participants: 35 HIV-1 infected men on ART (A); 35 HIV-1- infected men not on ART (B); and 34 HIV-1 uninfected men (C). RESULTS: The mean and standard deviation results for groups A, B, and C were 19.7 ± 6.56, 17.2 ± 3.96, and 16.9 ± 5.78 for ostase BAP; 7.9 ± 9.53, 8.5 ± 8.30, and 5.5 ± 1.65 for osteocalcin; and 3.9 ± 1.04, 3.1 ± 0.81, and 2.5 ± 0.59 for TRAP, respectively. Simple and multivariate analyses showed significant differences in mean TRAP and BAP concentrations between the three groups. In addition strong correlations between blood levels of Ostase BAP and TRAP (r=0.570, p=0.0004), and between blood levels of Ostase BAP and PTH (r=0.436, P=0.0098) for HIV-1 infected men on ART were observed. CONCLUSION: New strategies for measurement of blood and urine biochemical markers of bone formation and resorption during bone turnover can be useful for clinical monitoring of treatment of HIV-1 infected patients. Recently developed methods for measuring serum levels of TRAP and Ostase BAP represent superior laboratory tools for assessing the hyperactivity of osteoclasts, osteoblasts and bone loss in HIV-1 infected individuals receiving ART. Measurements of TRAP and BAP as bone turnover biomarkers are economical and are important for monitoring bone metabolism during ART and the need for osteoporosis treatment.

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.