Comparison of normalization methods for measuring immune markers in cervical secretion specimens

Marks, Morgan A.; Eby, Yolanda; Howard, Roslyn; Gravitt, Patti E.

doi:10.1016/j.jim.2012.05.012

Cited by 22 publications

(25 citation statements)

References 12 publications

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“…We examined the reproducibility of total protein (BCA, Thermo Fisher Scientific Inc., Rockford, IL), which has been used to normalize the levels of immune markers in cervical secretions [23], and albumin (Bethyl Laboratories, Inc., Montgomery, TX). The bile samples were diluted 1:100 for total protein, and 1:75000 for albumin.…”

Section: Methodsmentioning

confidence: 99%

Application of multiplex arrays for cytokine and chemokine profiling of bile

et al. 2015

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Background Gallbladder disease is highly related to inflammation, but the inflammatory processes are not well understood. Bile provides a direct substrate in assessing the local inflammatory response that develops in the gallbladder. To assess the reproducibility of measuring inflammatory markers in bile, we designed a methods study of 69 multiplexed immune-related markers measured in bile obtained from gallstone patients. Methods To evaluate assay performance, a total of 18 bile samples were tested twice within the same plate for each analyte, and the 18 bile samples were tested on two different days for each analyte. We used the following performance parameters: detectability, coefficient of variation (CV), intraclass correlation coefficient (ICC), and percent agreement (concordance among replicate measures above and below detection limit). Furthermore, we examined the association of analyte levels with gallstone characteristics such as type, numbers, and size. Results All but 3 analytes (Stem Cell Factor, SCF; Thrombopoietin, TPO; sIL-1RI) were detectable in bile. 52 of 69 (75.4%) analytes had detectable levels for at least 50% of the subjects tested. The within-plate CVs were ≤25% for 53 of 66 (80.3%) detectable analytes, and across-plate CVs were ≤25% for 32 of 66 (48.5%) detectable analytes. Moreover, 64 of 66 (97.0%) analytes had ICC values of at least 0.8. Lastly, the percent agreement was high between replicates for all of the analytes (median; within plate, 97.2%; across plate, 97.2%). In exploratory analyses, we assessed analyte levels by gallstone characteristics and found that levels for several analytes decreased with increasing size of the largest gallstone per patient. Conclusions Our data suggest that multiplex assays can be used to reliably measure cytokines and chemokines in bile. In addition, gallstone size was inversely related to the levels of select analytes, which may aid in identifying critical pathways and mechanisms associated with the pathogenesis of gallbladder diseases.

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Section: Methodsmentioning

confidence: 99%

Application of multiplex arrays for cytokine and chemokine profiling of bile

et al. 2015

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“…Prior work conducted by our group demonstrated a strong correlation of immune marker concentration with specimen volume [18]. Therefore, to minimize sampling bias, immune marker concentrations were normalized to the concentration of total protein present in the extracted secretion specimen.…”

Section: Methodsmentioning

confidence: 99%

Comparison of the immune microenvironment of the oral cavity and cervix in healthy women

et al. 2013

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Background Despite similar frequencies of exposure, the low prevalence of certain sexually transmitted infections such as Chlamydia, HPV and HIV-1 in the oral cavity relative to the cervix is poorly understood. This could be explained in part by differences in host immune microenvironments between these two anatomic sites. Objective We compared the concentration and correlation of 27 different immune markers in paired secretion specimens collected from the oral and cervical mucosa of healthy women. Methods Paired oral and cervical secretion specimens were collected from thirty-nine women. The concentration of twenty-seven different immune markers was estimated using a Luminex multiplex assay. Marker concentration was normalized to total protein present in the specimen. Median immune marker concentrations were compared across anatomic sites. Unsupervised hierarchical clustering analysis was utilized to identify groups of markers that shared similar patterns of relative concentrations across anatomic sites. Results The oral cavity had significantly higher concentrations of eotaxin, IFN-γ, IL-2, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15, PDGF-BB, TNF-α, (p < 0.01 for each) while the cervix had higher concentrations of proinflammatory markers such as FGF-basic, IL-1ra, IL-1β, IL-6, IL-8, IP-10, G-CSF, GM-CSF, MCP-1, MIP-1β, VEGF (p < 0.01 for each). Hierarchical cluster analysis identified two groups of immune markers comprised of T-cell related immune markers with significantly higher concentrations in the oral cavity relative to the cervix, and a third cluster consisting of mostly inflammatory immune markers which were higher concentrations in the cervix. The oral cavity had a larger number of significant inter-marker correlations as compared to the cervix. Conclusions The oral cavity and cervix have significantly different immune marker profiles, which may in part explain the significantly lower burden of sexually transmitted infections such as Chlamydia, HPV, and HIV-1 in the oral cavity vs. the cervix.

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“…Normalization using total protein has been proposed as an alternative approach. 27 Thus, we also examined the results using total protein [(pg of immune markers)/(mg of total protein)] for normalization as a sensitivity analysis.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of a multiplex panel of immune‐related markers in cervical secretions: A methodologic study

Koshiol

Sklavos

Wentzensen

et al. 2013

Intl Journal of Cancer

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While persistent carcinogenic human papillomvirus (HPV) infection is necessary for cervical carcinogenesis, the co-factors involved in HPV persistence and disease progression are poorly understood. Chronic cervical inflammation may increase risk, but few studies have measured immune markers (cytokines/chemokines/soluble receptors) in cervical secretions. We evaluated the performance of 74 multiplexed, bead-based immune markers in cervical secretions from three groups of women with biopsy evaluation of cervical intraepithelial neoplasia (CIN): 1) 25% detectability and >80% interclass correlation coefficients (ICC) acceptable for epidemiologic studies. Within-batch coefficients of variation (CV) of ≥25% indicated room for assay improvement. Secondarily, we explored associations between marker levels and CIN/HPV status adjusted for matching variables, assay batch, age, and number of sexual partners. Sixty-two markers (84%) had >25% detectability and ICCs>80%. Of those, 53 (85%) had CVs<25%. Using these preliminary data, we found that HPV-positivity was associated with increased eotaxin-1 (OR: 15.63, 95% CI: 1.26–200.00) and G-CSF (OR: 12.99, 95% CI: 1.10–142.86) among CIN-negative women. There was suggestive evidence that higher chemoattractant marker levels were associated with CIN2/3 (e.g., MIP-1delta, OR: 4.48, 95% CI: 0.87–23.04 versus

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Comparison of normalization methods for measuring immune markers in cervical secretion specimens

Cited by 22 publications

References 12 publications

Application of multiplex arrays for cytokine and chemokine profiling of bile

Application of multiplex arrays for cytokine and chemokine profiling of bile

Comparison of the immune microenvironment of the oral cavity and cervix in healthy women

Evaluation of a multiplex panel of immune‐related markers in cervical secretions: A methodologic study

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