Application of multiplex arrays for cytokine and chemokine profiling of bile.
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.
HPV Immunology Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: kemptj@mail.nih.gov.
Troy J
TJ
Kemp
Infections and Immunoepidemiology Branch, DCEG, NCI, NIH, Bethesda, MD, USA. Electronic address: felipe.castro@nih.gov.
Felipe A
FA
Castro
Department of Epidemiology, Shanghai Cancer Institute, Shanghai Cancer Institute, Shanghai, China. Electronic address: ytgao@vip.sina.com.
Yu-Tang
YT
Gao
Infections and Immunoepidemiology Branch, DCEG, NCI, NIH, Bethesda, MD, USA. Electronic address: hildesha@exchange.nih.gov.
Allan
A
Hildesheim
Infections and Immunoepidemiology Branch, DCEG, NCI, NIH, Bethesda, MD, USA. Electronic address: leticia.nogueira@nih.gov.
Leticia
L
Nogueira
Department of General Surgery, Zhongshan Hospital, School of Medicine, Fudan University, Shanghai, China. Electronic address: wang.bingsheng@zs-hospital.sh.cn.
Bing-Sheng
BS
Wang
Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China. Electronic address: sunlush@126.com.
Lu
L
Sun
HPV Immunology Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: Gloriana.Shelton@nih.gov.
Gloriana
G
Shelton
Biostatistics Branch, DCEG, NCI, NIH, Bethesda, MD, USA. Electronic address: pfeiffer@mail.nih.gov.
Ruth M
RM
Pfeiffer
Cancer Prevention Institute of California, Fremont, CA, USA; Stanford Cancer Institute, Palo Alto, CA, USA; Department of Health Research and Policy, School of Medicine, Stanford University, Palo Alto, CA, USA. Electronic address: Ann.Hsing@cpic.org.
Ann W
AW
Hsing
HPV Immunology Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: pintol@mail.nih.gov.
Ligia A
LA
Pinto
Infections and Immunoepidemiology Branch, DCEG, NCI, NIH, Bethesda, MD, USA. Electronic address: koshiolj@mail.nih.gov.
Jill
J
Koshiol
D000328:Adult; D000368:Aged; D001646:Bile / Q000378:metabolism*; D018925:Chemokines / Q000378:metabolism*; D002769:Cholelithiasis; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results
D018925:Chemokines
Bile; Chemokines; Cytokines; Gallstones