| Background and objective:Symptoms of early primary hepatic carcinoma(PHC)are not typical.The presence of typical symptoms usually indicates the cancer at advanced stage and the optimal timing for treatment has lost.Therefore the early diagnosis of PHC is very important.Serum alpha-fetoprotein(AFP)detection and medical imaging examination are the primary tools for clinical diagnosis of PHC,but they are not ideal for small primary hepatic cancer.Aptamers are synthetic single-stranded oligonucleotides with advantages over antibodies in binding targets with high affinity and specificity,and thus show a good prospect in the diagnosis of diseases,especially tumors.In previous work,we successfully selected a group of aptamers against PHC serum through the SELEX technique,established a new method based on triple fluorescence analysis for aptamer application in diagnostics,and found that the aptamers showed various diagnostic values in different experimental conditions.In the present study,we will assess the diagnostic values of different combinations of experimental conditions to provide foundation for the establishment of a new diagnostic method for PHC based on aptamers.Methods:1.Collection of serum specimens and the related clinical data: The serum specimens before therapy were collected from the patients with PHC(n=104)or liver cirrhosis(LC)(n=104)hospitalized in the First Affiliated Hospital of Nanchang University during 2014 to 2015 and frozen at-80°C.The available clinical data of these patients were also collected at the same time,mainly including age,gender,and the results of serum tumor markers,hepatic function tests,medical imaging examination and pathology.2.Serum fluorescence intensities were measured before and after adding the aptamers: PHC serum aptamers AP-HCS-11-6,AP-HCS-9-89 were synthesized.The triple serum fluorescence intensities(autofluorescence,Eva Green-related fluorescence and aptamer-related fluorescence)were measured through a real-time PCR instrument.Six experimental conditions for triple fluorescence detection were set up through the different combinations of serum 3μL,2×Eva Green 2.5 or 5μL and aptamers 0.3,0.5 or 0.7 pmol.the fluorescence intensities of each serum specimen were measured in 8,18,28,37,47 and 55℃ under each condition.3.Diagnostic evaluation of the fluorescence indicators for PHC: Fluorescence indicators were analyzed by the receiver operating characteristic(ROC)curve and their diagnostic values were evaluated by the area under ROC curve(AUROC)and diagnostic performances(sensitivity,specificity,accuracy,negative predictive value,positive predictive value,negative likelihood ratio and positive likelihood ratio)according to ROC curve analysis.The diagnostic value of a single fluorescence indicator was directly evaluated by ROC curve.For the evaluation of diagnostic value of multiple fluorescence indicators combined,a diagnostic model was established by the binary Logistic regression analysis and the predicted group probability of the model for specimens was used as the test variable for ROC curve analysis.4.Diagnostic evaluation of combination analysis for PHC: Diagnostic values of combination analyses of the results detected at different conditions or temperatures for PHC were evaluated by the same methods as above,including single aptamer-based detection(single condition under each temperature point,double conditions under each temperature point,triple conditions under each temperature point,double temperatures under each condition,and triple temperatures under each condition),double aptamer-based detection(single condition under each temperature point,double conditions under each temperature point,and double temperatures under each condition),and the combination of aptamers with AFP.Results:1.Diagnostic values of single aptamer-based combination analysis for PHC:Diagnostic values of fluorescence indicators detected under different temperatures or experimental conditions were different between aptamers AP-HCS-11-6 and AP–HCS-9-89.The highest AUROC of single fluorescence indicators was 0.721.Combination analyses of multiple fluorescence indicators were much more valuable than any single indicator for diagnosing PHC,with the highest AUROCs more than0.9 at the combinations of double and triple conditions under different temperatures and double and triple temperatures under different conditions.2.Diagnostic values of double aptamer-based combination analysis for PHC:The diagnostic values of double aptamer-based combination analyses were superior to single aptamer for diagnosing PHC,with the highest AUROCs more than 0.9 at the combinations of double conditions under different temperatures and double temperatures under different conditions.3.Diagnostic values of the combination of aptamers with AFP for PHC: The AUROC,specificity,sensitivity,accuracy were all higher than 0.9 in combination analyses of fluorescence indicators of two aptamers detected at double temperatures under the same condition combined with AFP,better than other types of combination analyses.Conclusions:1.The values of aptamers against PHC serum for diagnosing PHC are different in various temperatures and experimental conditions.2.Combination analyses of single aptamer-based results detected at different temperatures and conditions are able to effectively improve the diagnostic value for PHC.3.Two aptamer-based combination analysis is more valuable for diagnosing PHC than any single aptamer.4.The combination of aptamers with AFP can improve the diagnostic values for the diagnosis of PHC. |
