A digital quantification method for the detection of biomarkers on a microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.

472 Background: Alpha-fetoprotein (AFP) is the most widely used biomarker for hepatocellular carcinoma (HCC) prognosis. However, AFP is not useful in establishing a prognosis for patients with a tumor in the early stages. hPG80 (circulating progastrin) is a tumor promoting peptide present in the blood of patients with various cancers including HCC, even at early stages. In this study, we evaluated the prognostic value of plasma hPG80 in patients with HCC at early and intermediate stages. Methods: A total of 79 HCC patients including 44 BCLC from 0 to A and 35 BCLC B managed with local or systemic treatments, (“Liverpool” biobank) were enrolled prospectively and analyzed retrospectively. hPG80 was quantified using DxPG80 Lab kit (ECS-Progastrin) with a limit of detection at 1 pM and AFP was quantified using Cobas E411 in the blood of HCC patients. An optimal cutoff value of hPG80 was identified at 4.5 pM by calculating the minimal p-value based on the log-rank method. For AFP, a cutoff of 100 ng/mL was used as for liver transplantation (Notarpaolo, 2016). The prognostic impact of hPG80 and AFP levels on patient overall survival (OS) was assessed using Kaplan-Meier curves and log-rank tests. Results: hPG80 was detected in 36 of 44 (81.8%) HCC patients at stages BCLC 0 to A and 29 of 35 (82.9%) at stage BCLC B by contrast to AFP present in only 5 (11.4%) and 9 (25.7%) patients respectively. The median of hPG80 was 5.7 pM (IQR: 1.7-22.4 pM) at stages BCLC 0 to A, 6.0 pM (IQR: 1.5-22.2 pM) at stage BCLC B and significantly different from a non-HCC age-range matched control group cohort (median value: 1.5 pM, IQR 0.6-3.2 pM, p < 0.0001). When combining BCLC 0 to B, the median OS for hPG80+ patients (n = 46) was significantly shorter than that of hPG80- patients (n = 33) (25 months versus undefined, HR: 3.07, 95% CI: 1.46-6.43; p = 0.0064). AFP+ patients were also significantly correlated with poorer OS (17.5 months versus undefined, HR: 3.16, 95% CI: 1.06-9.47; p = 0.0030). Then, we evaluated the prognostic significance of hPG80 in combination with AFP levels. hPG80+ patients with AFP > 100 ng/mL (n = 46) had a worse OS than that of patients with AFP < 100 ng/mL patients (n = 33) (15.8 months versus undefined, HR: 6.38, 95% CI: 1.74-23.41; p = 0.0052). Conclusions: Our findings show that hPG80 could serve as a new prognostic biomarker for HCC patients at early to intermediate stages. These results will need to be confirmed in a prospective study, but it opens the possibility to use hPG80 as a biomarker for HCC patients at early stage at a time they can be treated to be cured.

Alpha fetoprotein (AFP) is the most widely used diagnostic and prognostic serum biomarker for hepatocellular carcinoma (HCC). Despite its wide clinical use, a systematic clinicopathologic study comparing AFP expression in HCC in situ with serum AFP concentrations has not yet been conducted. To analyze AFP expression in a large cohort of patients by immunohistochemistry, we employed a comprehensive tissue microarray with 871 different HCCs of overall 561 patients. AFP immunoreactivity was detected in only about 20% of HCC core biopsies, whereas 48.9% of the patients displayed increased serum values (>12 ng/mL). Immunostaining of whole tumor slides revealed that lack of detectable immunoreactivity in core biopsies in a subgroup of patients with elevated AFP serum concentrations is due to heterogeneous intratumoral AFP expression. Serum AFP concentrations and AFP expression in situ were moderately correlated (Spearman's rank correlation coefficient .53, P=1.2e - 13). High AFP expression detected in serum (>227.3ng/mL) or in situ predicted unfavorable prognosis and was associated with vascular invasion, higher tumor grade and macrotrabecular-massive tumor subtype. Multivariate and ROC curve analysis demonstrated that high AFP concentrations in serum is an independent prognostic parameter and represents the more robust prognostic predictor in comparison to AFP immunostaining of core biopsies. The previously published vessels encapsulating tumor clusters (VETC) pattern turned out as an additional, statistically independent prognostic parameter. AFP-positivity was associated with increased tumor cell apoptosis, but not with increased vascular densities. Additionally, AFP-positive tumors displayed increased proliferation rates, urea cycle dysregulation and signs of genomic instability, which may constitute the basis for their increased aggressiveness.

Binding of 16α-[ 18F]Fluoro-17β-estradiol to alphafetoprotein in Sprague-Dawley female rats affects blood levels

A Fe3O4@Au-basedpseudo-homogeneous electrochemical immunosensor for AFP measurement using AFP antibody-GNPs-HRP as detection probe

Plasma alphafetoprotein (AFP) concentrations are high at birth and decline progressively to reach adult levels between eight and 24 months of age. AFP is an important tumour marker for hepatoblastoma but may also increase following hepatic resection. The liver and intestine have similar embryonic origins from endoderm, so we postulated that plasma AFP may also rise after extensive bowel resection. AFP was measured postoperatively in plasma in four infants who had undergone resections of large amounts of intestine. AFP was measured twice in two babies and multiple times in the other two. In three of the four infants, the AFP concentrations were markedly above the expected levels for age, and in all four babies, AFP concentrations rose when they may have been expected to fall. Rising plasma AFP concentrations post extensive bowel resection in infants is a new finding which is possibly due to intestinal regeneration.

Same-day confirmation of intrauterine pregnancy failure in women with first- and early second-trimester bleeding

Alpha-fetoprotein in human fetal cerebrospinal fluid

253 Background: High alpha-fetoprotein (AFP) is associated with worse survival in hepatocellular carcinoma (HCC) patients. CA19-9 is a glycoprotein biomarker of biliary and pancreatic cancer. To our knowledge, no prospective study has examined CA19-9 as a prognostic biomarker in HCC. We hypothesized that it may add to our ability to risk stratify patients. Methods: We prospectively enrolled 122 patients with newly diagnosed HCC between 10/2008 and 7/2012 and followed until July 23, 2012. HCC was defined by AASLD criteria. CA 19-9 and AFP were measured on enrollment: 50% patients had CA19-9 >37 U/mL and 22% had CA 19-9>100. Pathology specimens were available for 50% of patients and none were consistent with mixed HCC-cholangiocarcinoma (HCC-CC) or pure CC. We evaluated the relationship between CA19-9 and overall survival using Kaplan Meier curves, univariate, and multivariate Cox Proportional Hazards Models. Results: The mean age of the cohort was 62.1 and 79% were male. 59% had underlying HCV and 16% had HBV. In univariate analysis, CA19-9>100 predicted worse survival (HR=3.44, 95% CI: 1.79-6.61, p=0.0002). Other cut-points for CA19-9 yielded similar results, although not all were significant. In a multivariate model including CP Score (B vs. A), Milan (outside vs. within), and AFP (>100 vs. ≤100), CA19-9 >100 remained an independent risk factor for increased mortality (HR 3.95, 95% CI: 1.95-7.97, p=0.0001). CP score and Milan status had HRs of 2.57 (95% CI: 1.31-5.02, p=0.006) and 5.27 (95% CI: 2.05-13.57 p=0.0006). AFP was not an independent risk factor for worse survival with a HR of 1.69 (95% CI: 0.85-3.37, p=0.14). We also looked at models using different cut-points for AFP; CA19-9 added additional risk stratification in all models. Among patients with AFP>100, median survival was 15.5 months vs. 3.8 months when comparing those with CA19-9 ≤100 with CA19-9>100 (p=0.01, log-rank). Conclusions: CA 19-9 is a readily available biomarker that may be a novel predictor of survival in HCC patients. In our multivariate model, CA 19-9>100 almost quadrupled mortality risk. It might be particularly useful to risk stratify patients with normal AFP and those with very high AFP being considered for transplant.

Hepatocellular carcinoma (HCC) is an aggressive tumor with agrowing socioeconomic burden. International guidelines do not predominantly recommend the pretherapeutic determination of the alpha-fetoprotein (AFP) concentration. Regarding the prognostic value of AFP, the European study data are not sufficiently meaningful. This study aimed to demonstrate possible aspects of the AFP level and to investigate the prognostic value of AFP levels as well as to provide impetus for future prospective studies. At the time of data retrieval the prospective liver databank showed 1382 entries. All patients with ahistologically confirmed HCC were included resulting in 92final entries. For these patients, information on T, N, M and G stages, R status as well as sex, age and etiology of the HCC were available. For data analysis the patient population was divided into six groups based on three cut-off values. Furthermore, asurvival analysis was performed using Kaplan-Meier and amultifactorial analysis of the influencing factors regarding outcome. The AFP serum level showed a statistically significant correlation with the tumor diameter (T1/T2 vs. T3/T4) and grading (G1/G2 vs. G3/G4). The survival prognosis was significantly lower in patients with higher AFP values (p < 0.05). The median survival time for patients with AFP levels >8 μg/l was 35months, with AFP levels >200 μg/l or >400 μg/l showed areduced median survival of 15months and 11months, respectively. High AFP levels were a significant influencing factor for the outcome independent of the T stage, age and R status of patients in comparison to low AFP levels. Taking the present results into consideration, the AFP level can have a therapeutic usefulness. Therapeutic consequences could be derived from the height of the measured AFP concentration, with respect to the treatment strategy. Therefore, preoperative and postoperative determination of the AFP serum level is recommended in all HCC patients.

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies. Alpha-fetoprotein (AFP) has some limitations in diagnosing early HCC. Recently, long noncoding RNAs (lncRNAs) showed great potential as tumor diagnostic biomarkers, and lnc-MyD88 was previously identified as a carcinogen in HCC. Here, we explored its diagnostic value as a plasma biomarker. Quantitative real-time PCR was adopted to detect lnc-MyD88 expression in plasma samples of 98 HCC patients, 52 liver cirrhosis (LC) patients, and 105 healthy people. The correlation between lnc-MyD88 and clinicopathological factors was analyzed through chi-square test. The receiver operating characteristic (ROC) curve was used to analyze the sensitivity, specificity, Youden index, and area under the curve (AUC) of lnc-MyD88 and AFP alone and in combination for the diagnosis of HCC. The relationship between MyD88 and immune infiltration was analyzed by single sample gene set enrichment analysis (ssGSEA) algorithm. Lnc-MyD88 was highly expressed in plasma samples of HCC and hepatitis B virus (HBV)-associated HCC patients. Lnc-MyD88 had better diagnostic value than AFP in HCC patients using healthy people or LC patients as control (healthy people, AUC: 0.776 vs. 0.725; LC patients, AUC: 0.753 vs. 0.727). The multivariate analysis showed that lnc-MyD88 had great diagnostic value for distinguishing HCC from LC and healthy people. Lnc-MyD88 had no correlation with AFP. Lnc-MyD88 and AFP were independent diagnostic factors for HBV-associated HCC. The AUC, sensitivity, and Youden index of the combined diagnosis of lnc-MyD88 and AFP combined were higher than those of lnc-MyD88 and AFP alone. The ROC curve of lnc-MyD88 for the diagnosis of AFP-negative HCC was plotted with a sensitivity of 80.95%, a specificity of 79.59%, and an AUC value of 0.812 using healthy people as control. The ROC curve also presented its great diagnostic value using LC patients as control (sensitivity: 76.19%, specificity: 69.05%, AUC value: 0.769). Lnc-MyD88 expression was correlated with microvascular invasion in HBV-associated HCC patients. MyD88 was positively correlated with infiltrating immune cells and immune-related genes. The high expression of plasma lnc-MyD88 in HCC is distinct and could be utilized as a promising diagnostic biomarker. Lnc-MyD88 had great diagnostic value for HBV-associated HCC and AFP-negative HCC, and it had higher efficacy in combination with AFP.

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A novel aptasensor based on light-addressable potentiometric sensor for the determination of Alpha-fetoprotein

In this paper, we present an integrated and automated prototype system which has been developed for real-time polymerase chain reaction (PCR) analysis based on microfluidic PCR array chips. The system integrates the PCR thermal cycling and optical detection capabilities to enable real-time fluorescence imaging and image processing for data analysis. The main advantage of the system is that it provides a solution that can rapidly perform and evaluate PCR experiment simultaneously on microfluidic PCR array chips. The system has demonstrated fast and efficient on-chip real-time PCR analysis using human genomic DNA samples. The implementation of the system integration is a multi-thread Windows software with component structure which is written in Visual C++.

This study aimed to identify novel long noncoding RNA (lncRNA) biomarkers for hepatocellular carcinoma (HCC) using publicly available tissue genomic datasets and validate their diagnostic utility for early‐stage HCC. Differentially expressed lncRNAs between 371 HCC and 50 nontumor tissues were obtained from The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC) project. Subsequently, the expression of the serum‐ and extracellular vesicle (EV)‐derived lncRNA was assessed in 10 patients with HCC and 10 healthy controls using RT–qPCR. The candidate lncRNAs were validated in 90 HCC and 92 non‐HCC (29 healthy control, 28 chronic hepatitis, 35 liver cirrhosis) patients. The sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) were calculated for the candidate lncRNAs and the current HCC biomarker, alpha‐fetoprotein (AFP). SFTA1P, HOTTIP, HAGLROS, LINC01419, HAGLR, CRNDE, and LINC00853 were markedly upregulated in HCC in TCGA_LIHC dataset. Among them, LINC00853 has not been reported in relation to HCC before. In patients with HCC, only expression of small EV‐derived LINC00853 (EV‐LINC00853) was increased. EV‐LINC00853 showed excellent discriminatory ability in the diagnosis of all‐stage HCC (AUC = 0.934, 95% confidence interval = 0.887–0.966). Moreover, using a 14‐fold increase and 20 ng·mL−1 as cutoffs for EV‐LINC00853 expression and AFP level, respectively, EV‐LINC00853 was found to have a sensitivity of 93.75% and specificity of 89.77%, while AFP showed only 9.38% sensitivity and 72.73% specificity for the diagnosis of early‐stage HCC (mUICC stage I). EV‐LINC00853 had a positivity of 97% and 67% in AFP‐negative and AFP‐positive early HCC, respectively. Serum EV‐derived LINC00853 may be a novel potential diagnostic biomarker for early HCC, especially for AFP‐negative HCC.