Serum Levels of α-Fetoprotein Increased More Than 10 Years Before Detection of Hepatocellular Carcinoma.

BACKGROUND & AIMS: Ultrasound (US)-based screening has been recommended for patients with an increased risk of hepatocellular carcinoma (HCC). US analysis, however, is limited in patients who are obese or have small tumors. The addition of serum level of α-fetoprotein (AFP) measurements to US analysis can increase detection of HCC. We analyzed data from patients with chronic liver disease, collected over 15 years in an HCC surveillance program, to develop a model to assess risk of HCC. METHODS: We collected data from 3450 patients with chronic liver disease undergoing US surveillance in Japan from March 1998 through April 2014, and followed them up for a median of 8.83 years. We performed longitudinal discriminant analysis of serial AFP measurements (median number of observations/patient, 56; approximately every 3 months) to develop a model to determine the risk of HCC. We validated the model using data from 2 cohorts of patients with chronic liver disease in Japan (404 and 2754 patients) and 1 cohort in Scotland (1596 patients). RESULTS: HCC was detected in 413 patients (median tumor diameter, 1.8 cm), during a median follow-up time of 6.60 years. In the development data set, the model identified patients who developed HCC with an area under the curve of 0.78; it correctly identified 74.3% of patients who did develop HCC, and 72.9% of patients who did not. Overall, 73.1% of patients were classified correctly. The model could be used to assign patients to a high-risk group (27.5 HCCs/1000 patient-years) vs a low-risk group (4.9 HCCs/1000 patient-years). A similar performance was observed when the model was used to assess patients with cirrhosis. Analysis of the validation cohorts produced similar results. CONCLUSIONS: We developed and validated a model to identify patients with chronic liver disease who are at risk for HCC based on change in serum AFP level over time. The model could be used to assign patients to high-risk vs low-risk groups, and might be used to select patients for surveillance.

Impact of disease stage and aetiology on survival in hepatocellular carcinoma: implications for surveillance.

BACKGROUND: Variation in survival in hepatocellular carcinoma (HCC) has been attributed to different aetiologies or disease stages at presentation. While international guidelines recommend surveillance of high-risk groups to permit early diagnosis and curative treatment, the evidence that surveillance decreases disease-specific mortality is weak. METHODS: We compared HCC survival figures from Japan (n=1174) and Hong Kong (n=1675) over similar time periods (Japan 2000-2013, Hong Kong, China 2003-2014). The former has an intensive national surveillance programme, while the latter has none. We also analysed changes in survival in Japan over a 50-year period including data from before and after institution of a national HCC surveillance programme. RESULTS: In Japan, over 75% of cases are currently detected by surveillance, whereas in Hong Kong <20% of cases are detected presymptomatically. Median survival was 52 months in Japan and 17.8 months in Hong Kong; this survival advantage persisted after allowance for lead-time bias. Sixty-two per cent of Japanese patients had early disease at diagnosis and 63% received curative treatment. The comparable figures for Hong Kong were 31.7% and 44.1%, respectively. These differences could not be accounted for by disease aetiology, and patients in Hong Kong who were detected at an early stage had a similar survival to the analogous patients in Japan. CONCLUSIONS: The variation in survival is largely accounted for by stage at diagnosis, which in turn relates to the intensity of surveillance programmes and the consequent variation in curative therapeutic options.

Role of the GALAD and BALAD-2 Serologic Models in Diagnosis of Hepatocellular Carcinoma and Prediction of Survival in Patients.

BACKGROUND & AIMS: GALAD and BALAD-2 are statistical models for estimating the likelihood of the presence of hepatocellular carcinoma (HCC) in individual patients with chronic liver disease and the survival of patients with HCC, respectively. Both models use objective measures, particularly the serum markers α-fetoprotein (AFP), AFP-L3, and des-γ-carboxyprothrombin. We aimed to validate these models in an international cohort of patients with HCC and assess their clinical performance. METHODS: We collected data on cancer diagnosis and outcomes of 6834 patients (2430 with HCC and 4404 with chronic liver disease) recruited from Germany, Japan, and Hong Kong. We also collected data from 229 patients with other hepatobiliary tract cancers (cholangiocarcinoma or pancreatic adenocarcinoma) and 92 healthy individuals (controls). For reference, the original UK cohort (on which the GALAD model initially was built and BALAD-2 was validated) was included in the analysis. We assessed the effects of tumor size and etiology on GALAD model performance, and its ability to correctly discriminate HCC from other hepatobiliary cancers. We assessed the performance of BALAD-2 in patients with different stages of HCC. RESULTS: In all cohorts, the area under the receiver operating characteristic curve (AUROC), quantifying the ability of GALAD to discriminate patients with HCC from patients with chronic liver disease, was greater than 0.90-similar to the series on which the model originally was built (AUROC, 0.97). GALAD discriminated patients with HCC from those with other hepatobiliary cancers with an AUROC value of 0.95; values were slightly lower for patients with small unifocal HCCs, ranging from 0.85 to 0.95. Etiology and treatment of chronic viral hepatitis had no effect on the performance of this model. BALAD-2 analysis assigned patients with HCC to 4 distinct prognostic groups-overall and when patients were stratified according to disease stage. CONCLUSIONS: We validated the performance of the GALAD and BALAD-2 models for the diagnosis of HCC and predicting patient survival, respectively (based on levels of the serum markers AFP, AFP-L3, and des-γ-carboxyprothrombin), in an international cohort of almost 7000 patients. These systems might be used in HCC surveillance and determination of patient prognosis.

On-chip quantitative PCR using integrated real-time detection by capillary electrophoresis.

Quantitative PCR (qPCR) has been widely used for the detection and monitoring of a variety of infectious diseases. PCR and CE were integrated into a microfluidic chip that was designed to achieve rapid real-time amplicon sampling, separation, and quantitation without requiring various probes. A novel chip design allows the overlapped execution of PCR and CE, minimizing the time required for CE analysis after each PCR cycle. The performance of the on-chip qPCR method was demonstrated using a 45-minutes model assay protocol for the phiX174 bacteriophage, and the multiplexing capability of the method was demonstrated by adding a second target, E. coli genomic DNA, to the model assay. The results indicate good sensitivity, reproducibility, and linearity over the tested assay range, 50 to 2 × 10(4) copies/25 µL reaction. Based on this performance, the on-chip qPCR method should be applicable to a wide variety of infectious disease detection and monitoring assays with the addition of suitable sample preparation protocols.

Biomarkers for Hepatocellular Carcinoma (HCC): An Update.

The past decades have witnessed increased use of biomarkers in disease management. A biomarker is any characteristic that can be objectively measured and evaluated as an indicator of normal biological process, pathogenic process, or pharmacological response to a therapeutic intervention. The clinical measurements of biomarkers can be carried out in vivo using imaging modalities like ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI), as well as in vitro utilizing serum or plasma or other body fluids as specimens. In contrast to the imaging modalities, a prominent value of serum biomarkers is that they could be biologically relevant and disease-specific to pathophysiologic or pathologic process of disease development. This article provides an update of serum biomarkers for hepatocellular carcinoma (HCC) in risk assessment for early detection through surveillance.

Combinations of biomarkers and Milan criteria for predicting hepatocellular carcinoma recurrence after liver transplantation.

Growing evidence suggests that pretransplant alpha-fetoprotein (AFP) predicts outcomes of hepatocellular carcinoma (HCC) patients treated with liver transplantation. We aimed to determine whether pretransplant AFP, Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3), and des-gamma-carboxyprothrombin (DCP) predicted HCC recurrence after transplantation. A retrospective cohort study of 313 HCC patients undergoing transplantation between 2000 and 2008 was conducted, and 48 (15.3%) developed recurrence during a median follow-up of 90.8 months. The 127 patients with available serum drawn before transplantation were included; they included 86 without recurrence and 41 with recurrence. Serum was tested for AFP, AFP-L3%, and DCP in a blinded fashion with the µTASWako i30 immunoanalyzer. All biomarkers were significantly associated with HCC recurrence. The hazard ratios (HRs) were 3.5 [95% confidence interval (CI), 1.9-6.7; P < 0.0001] for DCP ≥ 7.5 ng/mL and 2.8 (95% CI, 1.4-5.4; P = 0.002) for AFP ≥ 250 ng/mL. The HR increased to 5.2 (95% CI, 2.3-12.0; P < 0.0001) when AFP ≥ 250 ng/mL and DCP ≥7.5 ng/mL were considered together. When they were combined with the Milan criteria, the HR increased from 2.6 (95% CI, 1.4-4.7; P = 0.003) for outside the Milan criteria to 8.6 (95% CI, 3.0-24.6; P < 0.0001) for outside the Milan criteria and AFP ≥ 250 ng/mL and to 7.2 (95% CI, 2.8-18.1; P < 0.0001) for outside the Milan criteria and DCP ≥7.5 ng/mL. Our findings suggest that biomarkers are useful for predicting the risk of HCC recurrence after transplantation. Using both biomarkers and the Milan criteria may be better than using the Milan criteria alone in optimizing the decision of liver transplantation eligibility.

Clinicopathological Significance of Leucine-Rich α2-Glycoprotein-1 in Sera of Patients With Pancreatic Cancer.

OBJECTIVES: Leucine-rich α2-glycoprotein-1 (LRG-1) is an inflammatory protein. Serum LRG-1 levels can reportedly be used as a cancer biomarker for several types of carcinoma. In the present study, we investigated the clinical usefulness of serum LRG-1 levels as a biomarker of pancreatic cancer. METHODS: A total of 124 patients with pancreatic cancer, 35 patients with chronic pancreatitis (CP), and 144 healthy volunteers were enrolled in the study. Serum LRG-1 levels were assayed by enzyme-linked immunosorbent assay. Immunohistochemistry was used to examine LRG-1 expression in pancreatic cancer tissues. RESULTS: Serum LRG-1 levels were significantly increased in patients with pancreatic cancer compared with CP patients and healthy volunteers. The LRG-1 levels increased with progressive clinical stages of pancreatic cancer. Receiver operator curve analysis showed that a combination of carbohydrate antigen 19-9 and LRG-1 resulted in a higher area under the curve for the diagnosis of pancreatic cancer. Positive staining was observed in all cases of pancreatic cancer, but positive signal was scarcely detected in tissues from CP patients or normal surrounding tissue. CONCLUSIONS: These results suggest that serum LRG-1 is a promising biomarker for pancreatic cancer.

Assessment of liver function in patients with hepatocellular carcinoma: a new evidence-based approach-the ALBI grade.

PURPOSE: Most patients with hepatocellular carcinoma (HCC) have associated chronic liver disease, the severity of which is currently assessed by the Child-Pugh (C-P) grade. In this international collaboration, we identify objective measures of liver function/dysfunction that independently influence survival in patients with HCC and then combine these into a model that could be compared with the conventional C-P grade. PATIENTS AND METHODS: We developed a simple model to assess liver function, based on 1,313 patients with HCC of all stages from Japan, that involved only serum bilirubin and albumin levels. We then tested the model using similar cohorts from other geographical regions (n = 5,097) and other clinical situations (patients undergoing resection [n = 525] or sorafenib treatment for advanced HCC [n = 1,132]). The specificity of the model for liver (dys)function was tested in patients with chronic liver disease but without HCC (n = 501). RESULTS: The model, the Albumin-Bilirubin (ALBI) grade, performed at least as well as the C-P grade in all geographic regions. The majority of patients with HCC had C-P grade A disease at presentation, and within this C-P grade, ALBI revealed two classes with clearly different prognoses. Its utility in patients with chronic liver disease alone supported the contention that the ALBI grade was indeed an index of liver (dys)function. CONCLUSION: The ALBI grade offers a simple, evidence-based, objective, and discriminatory method of assessing liver function in HCC that has been extensively tested in an international setting. This new model eliminates the need for subjective variables such as ascites and encephalopathy, a requirement in the conventional C-P grade.

Changes in highly sensitive alpha-fetoprotein for the prediction of the outcome in patients with hepatocellular carcinoma after hepatectomy.

We investigated changes in highly sensitive lens culinaris agglutinin A-reactive fraction of alpha-fetoprotein (hsAFP-L3) measured using a novel method and its predictive ability for prognosis in patients with hepatocellular carcinoma (HCC) who underwent curative hepatectomy, comparing to other HCC tumor markers, that is, AFP, des-gamma-carboxy prothrombin (DCP), and AFP-L3 measured with conventional method (cAFP-L3). AFP, DCP, and AFP-L3 including both cAFP-L3 and hsAFP-L3 were measured before and after curative hepatectomy in 187 patients. The percentage of patients with elevated tumor marker levels pre- and postoperatively was compared, and recurrence-free and overall survival rates were analyzed based on changes in tumor markers. The percentages of patients with elevated AFP, DCP, and cAFP-L3 decreased postoperatively. In contrast, the percentage of patients with elevated hsAFP-L3 did not decrease postoperatively. Both recurrence-free and overall survival rates were significantly lower in patients whose tumor marker levels remained elevated postoperatively than patients without tumor marker elevation postoperatively. Recurrence-free and overall survival rates of patients in whom hsAFP-L3 became elevated postoperatively despite normal preoperative hsAFP-L3 levels were significantly lower than those of patients with normal hsAFP-L3 postoperatively, and were similar to those of patients with persistent elevation. Preoperative elevations of AFP, DCP, and cAFP normalized in many patients postoperatively, but not for hsAFP-L3. The elevation of hsAFP-L3 identifies patients with poor prognosis despite the normalization of AFP and DCP.

The detection of hepatocellular carcinoma using a prospectively developed and validated model based on serological biomarkers.

BACKGROUND: Hepatocellular carcinoma is a common complication of chronic liver disease (CLD), and is conventionally diagnosed by radiological means. We aimed to build a statistical model that could determine the risk of hepatocellular carcinoma in individual patients with CLD using objective measures, particularly serological tumor markers. METHODS: A total of 670 patients with either CLD alone or hepatocellular carcinoma were recruited from a single UK center into a case-control study. Sera were collected prospectively and specifically for this study. A logistic regression analysis was used to determine independent factors associated with hepatocellular carcinoma and a model built and assessed in terms of sensitivity, specificity, and proportion of correct diagnoses. RESULTS: The final model involving gender, age, AFP-L3, α fetoprotein (AFP), and des-carboxy-prothrombin ("GALAD") was developed in a "discovery" data set and validated in independent data sets both from the same institution and from an external institution. When optimized for sensitivity and specificity, the model gave values of more than 0.88 irrespective of the disease stage. CONCLUSIONS: The presence of hepatocellular carcinoma can be detected in patients with CLD on the basis of a model involving objective clinical and serological factors. It is now necessary to test the model's performance in a prospective manner and in a routine clinical practice setting, to determine if it may replace or, more likely, enhance current radiological approaches. IMPACT: Our data provide evidence that an entirely objective serum biomarker-based model may facilitate the detection and diagnosis of hepatocellular carcinoma and form the basis for a prospective study comparing this approach with the standard radiological approaches.

High-sensitivity Lens culinaris agglutinin-reactive alpha-fetoprotein assay predicts early detection of hepatocellular carcinoma.

BACKGROUND: Prognosis of patients with hepatocellular carcinoma (HCC) remains poor because HCC is frequently diagnosed late. Therefore, regular surveillance has been recommended to detect HCC at the early stage when curative treatments can be applied. HCC biomarkers, including Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3), are widely used for surveillance in Japan. A newly developed immunoassay system measures AFP-L3 % with high sensitivity. This retrospective study aimed to evaluate clinical utility of high-sensitivity AFP-L3 (hs-AFP-L3) as a predictor of early stage HCC in surveillance at a single site. METHODS: Of consecutive 2830 patients in the surveillance between 2000 and 2009, 104 HCC-developed and 104 non-HCC patients were selected by eligibility criteria and propensity score matching. Samples were obtained from the HCC patients who had blood drawn annually for 3 years prior to HCC diagnosis. RESULTS: In the present study, hs-AFP-L3 was elevated 1 year prior to diagnosis in 34.3 % of patients. The survival rate of patients with the hs-AFP-L3 ≥ 7 % at 1 year prior to diagnosis was significantly lower than that of patients with hs-AFP-L3 < 7 %. CONCLUSIONS: Elevation of hs-AFP-L3 was early predictive of development of HCC even at low AFP levels and in absence of ultrasound findings of suspicious HCC. The hs-AFP-L3 should be added to surveillance programs with US because elevated hs-AFP-L3 may be a trigger to perform enhanced imaging modalities for confirmation of HCC.

Reference change values for lens culinaris agglutinin-reactive α-fetoprotein and des-γ-carboxy prothrombin in patients with chronic hepatitis C.

BACKGROUND: Lens culinaris agglutinin-reactive α-fetoprotein (AFP-L3) and des-γ-carboxy prothrombin (DCP) have been routinely used as serological tumor markers of hepatocellular carcinoma (HCC) for surveillance. The aims of this study were: (i) to determine the biological variation of AFP-L3 and DCP in patients with chronic hepatitis C; and (ii) to calculate the reference change values (RCVs) of AFP-L3 and DCP. METHODS: Ten patients with cirrhosis due to hepatitis C virus (HCV) infection and without HCC were enrolled in the study. Serum samples were collected at 14-day intervals, and 10 samples in total were obtained for each patient. AFP-L3 and DCP levels were measured by microchip capillary electrophoresis and liquid-phase binding assay. Intra-individual (CV(I)) and inter-individual (CV(G)) biological variations and RCVs were estimated from the data generated. RESULTS: The CV(I) was 29.0% for AFP-L3 and 24.6% for DCP, and CV(G) was 63.5% for AFP-L3 and 40.4% for DCP. The RCVs for AFP-L3 and DCP were 68.3% and 58.5%, respectively. CONCLUSIONS: Increases in values for AFP-L3 and DCP within 68.3% and 58.5% may be biological variations. Clinician should take these variations into consideration for the management of patients with HCV infection under surveillance of HCC.

Clinical utility of highly sensitive Lens culinaris agglutinin-reactive alpha-fetoprotein in hepatocellular carcinoma patients with alpha-fetoprotein <20 ng/mL.

The Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3) has been used as a diagnostic and prognostic marker of hepatocellular carcinoma (HCC). The analytical sensitivity of a conventional method for AFP-L3% is not sufficient in patients with a low AFP level. This study was performed to determine the clinical utility of a newly developed highly sensitive AFP-L3% (hs-AFP-L3%) assay in patients with an AFP level <20 ng/mL. In the cohort study, serum samples obtained from 270 patients with newly diagnosed HCC before treatment and 396 patients with chronic liver disease at Ogaki Municipal Hospital, in both of which the AFP level was <20 ng/mL, were measured for conventional AFP-L3% (c-AFP-L3%), hs-AFP-L3% and des-gamma-carboxy prothrombin (DCP). Diagnostic sensitivity and specificity of hs-AFP-L3% at a cut-off level of 5% were 41.5% and 85.1%, respectively, significantly increasing the sensitivity from 7.0% for c-AFP-L3%. Multivariate analysis identified hs-AFP-L3% as an independent factor associated with reduced long-term survival. The survival rate of patients with high hs-AFP-L3% (≥ 5%) before treatment was significantly poorer than that of patients with low hs-AFP-L3% (<5%) (P < 0.001). In patients with AFP <20 ng/mL, measurements of AFP-L3% by the highly sensitive method before treatment were more useful for diagnosis and prognosis of HCC than by the conventional method.

Clinical advantage of highly sensitive on-chip immunoassay for fucosylated fraction of alpha-fetoprotein in patients with hepatocellular carcinoma.

BACKGROUND: Alpha-fetoprotein (AFP) has been widely used as a diagnostic master for hepatocellular carcinoma (HCC), and the fucosylated fraction of AFP (AFP-L3) has been reported to be a specific marker for HCC. However, AFP-L3 has not always been reliable in cases with low serum AFP concentrations. Recently, a novel automated immunoassay for AFP-L3, the micro-total analysis system (µ-TAS), has been developed. AIM: The aim of this study is to evaluate the clinical usefulness of µ-TAS AFP-L3. METHODS: Serum AFP-L3 was measured in 295 patients with HCC and in 350 with benign liver diseases. The diagnostic accuracy of µ-TAS AFP-L3 was compared with that of the conventional assay (liquid-phase binding assay; LiBASys). The relationship between µ-TAS AFP-L3 and clinical features was investigated. RESULTS: When the cutoff value was set at 7%, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of µ-TAS AFP-L3 were 60.0%, 90.3%, 76.4%, 83.9%, and 72.8%, respectively. Its sensitivity was particularly good (41.1%) in HCC subgroups with lower AFP concentrations (<20 ng/ml). The positivity rates for µ-TAS AFP-L3 were higher at each tumor stage than those of LiBASys AFP-L3 (µ-TAS/LiBASys: stage I, 44.2%/16.3%; stage II, 52.9%/37.5%; stage III, 66.4%/44.5%; stage IV, 82.8%/65.5%). CONCLUSIONS: µ-TAS AFP-L3 is more sensitive for discriminating HCC than the conventional LiBASys AFP-L3, particularly in subgroups with lower AFP concentrations and early-stage HCC.

Clinical application of a lectin-antibody ELISA to measure fucosylated haptoglobin in sera of patients with pancreatic cancer.

BACKGROUND: Recent advanced techniques in glycobiology have produced a number of tumor marker candidates. As a result from the glycomic approach, we found that fucosylated haptoglobin in sera was a possible tumor marker for pancreatic cancer (PC). Although Aleuria aurantia lectin (AAL) blotting can detect fucosylated haptoglobin, it is difficult to quantify fucosylated haptoglobin precisely. To overcome this problem, we developed a fucosylated haptoglobin detection kit as a sandwich enzyme-linked immune sorbent assay (ELISA) using AAL and the Fab portion of anti-haptoglobin antibody. In the present study, we investigated the clinical application of this lectin-antibody ELISA kit to measure fucosylated haptoglobin in PC. METHODS: We measured fucosylated haptoglobin in patients with PC with a lectin-antibody ELISA kit. The fucosylated haptoglobin measured with this assay was compared with lectin blotting data, and the discrepancy was analyzed by immunoprecipitation methods. The concentration of fucosylated haptoglobin was investigated with respect to the clinical stage of PC. We also measured fucosylated haptoglobin, using 397 cases of several types of cancers including PC, benign diseases, and normal controls. RESULTS: The sensitivity and specificity for the differential diagnosis of PC from normal controls was 50% and 91%, respectively. The results from lectin-antibody ELISA were significantly correlated with data from previous AAL blotting studies. Positive rates of fucosylated haptoglobin with this method in patients with PC were significantly higher in cases of stage IV compared with other clinical stages. Fucosylated haptoglobin was increased in several types of cancers, in which fucosylated haptoglobin was reported to increase. CONCLUSIONS: While certain cases showed a discrepancy in fucosylated haptoglobin concentrations between the lectin-antibody ELISA and conventional lectin blotting, this novel type of lectin-antibody ELISA might be useful for a tumor marker for PC.

[Evaluation of a new, microfluidic chip-based immunoassay for measurement of AFP-L3].

PURPOSE: AFP-L3 is an isoform of a-fetoprotein which has a fucosylated carbohydrate chain, and the fraction of AFP-L3/total AFP (AFP-L3%) specifically increases in hepatocellular carcinoma (HCC) patients and is widely used for screening and prognosis of HCC. The newly developed microTAS method which combines microchip electrophoresis and lectin affinity electrophoresis can rapidly provide AFP-L3% and total AFP measurements simultaneously at higher sensitivity. Here, we evaluated the system to know its analytical performance and clinical utility. METHOD: Fully automated immunoanalyzer, microTASWako i30 which utilizes Liquid-phase Binding Assay-Electrokinetic Analyte Transport Assay (LBA-EATA method) as the assay principle was employed for the measurement of total AFP and AFP-L3%. We evaluated detection sensitivity, precision, accuracy, and correlation of the method. RESULTS: The detection sensitivity was 0.3 ng/ml for both AFP-L1 and L3. The accuracy of the assay was 91.3-105.0% for total AFP. The precision of the assay was CV 1.9% at 2 ng/ml of total AFP, and CV 1.3% for 10% of AFP-L3% at 20ng/ml of total AFP. The microTAS method showed good correlation with the lectin affinity electrophoresis (AFP-L3 Test Wako) and the LBA methods (LBA Wako AFP-L3 on LiBASys) methods, giving correlation coefficient (r) of 0.988 and 0.988, respectively. The microTAS immunoreaction assay time and the total assay time including chip preparation were 1 and 9 min, respectively. CONCLUSION: Since the microchip assay is rapid and highly sensitive, it should have better clinical utility than the current methods.

[Fully automated immunoassay system utilizing microchip electrophoresis].

Application of microTAS (micro Total Analysis Systems) technologies utilizing chips with microfluidic channels to clinical diagnostic testing has drawn a lot of attention since it is expected to contribute to shortening reaction time, reduction of reagent/sample consumption, reducing instrument size, and other advantages of microchip electrophoresis. We have developed a fully automated immunoassay system by employing isotachophoresis followed by capillary gel electrophoresis for immunoreaction and B/F separation in microfluidic channels on polymer microchips. Laser-Induced Fluorescence (LIF) was used for detection of the sandwich immunocomplex composed of DNA-conjugate antibody, antigen and fluorescent dye-conjugated antibody. An immunoassay for PIVKA II was demonstrated on this new microTAS system utilizing the DNA-conjugated anti PIVKA II antibody and the fluorescent-dye labeled anti-prothrombin antibody. The resulting assay showed good assay performance with high sensitivity (LOD = 5mAU/mL), good reproducibility(CV = 1.0 - 5.7%) and good correlation with the commercially available PIVKA II assay kit (regression curve of y = 1.04x + 11.1, r = 0.991). The assay turn around time (TAT) was about 9 min. The PIVKA II assay will be useful for the diagnosis and prognosis of hepatocellular carcinoma.

Urinary calreticulin in the diagnosis of bladder urothelial carcinoma.

OBJECTIVES: To evaluate the potential suitability of calreticulin (CRT) as a urinary marker for bladder cancer. METHODS: Urine specimens were collected from patients with histologically confirmed bladder urothelial carcinoma (Group 1; n = 109), urological patients without urothelial carcinoma (Group 2; n = 60), and non-urological patients (Group 3; n = 40). We developed an enzyme-linked immunosorbent assay (ELISA) procedure using commercially available anti-CRT mono/polyclonal antibodies, and then measured the concentration of urinary CRT. RESULTS: Urinary CRT concentration of group 1 was significantly higher than group 2 and 3 (Mann-Whitney U-test, P < 0.001). Groups 2 and 3 were joined together and considered as a non-bladder cancer group (n = 100), and a cutoff value (2.85 ng/mL) was determined using receiver operating characteristic (ROC) analysis. The sensitivity, specificity, and the area under the curve were 67.9%, 80.0%, and 0.742, respectively. The overall sensitivity of voided urine cytology (VUC) was 39.0% (n = 105), and the sensitivity of urinary CRT was significantly superior to VUC (McNemar test, P < 0.001). Higher sensitivity was observed especially in Ta, G1-2, and

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis.

Implementation of the on-chip immunoassay for alpha-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r(2)=0.981 and slope=1.03.

Utility of Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein and des-gamma-carboxy prothrombin, alone or in combination, as biomarkers for hepatocellular carcinoma.

BACKGROUND & AIMS: Des-gamma-carboxy prothrombin (DCP) and Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3) are surveillance markers used to detect hepatocellular carcinoma (HCC) in Japan. This study evaluated their utility, alone or in combination, in a North American population. METHODS: Patients with hepatitis C virus-related cirrhosis were followed up prospectively for 2 years. RESULTS: Of 372 patients, HCC developed in 34 of 298 who were free of HCC at entry. The overall sensitivity, specificity, and positive and negative predictive values for only AFP (>20 ng/mL) were 61%, 71%, 34%, and 88%, respectively; for only AFP-L3% (>10) were 37%, 92%, 52%, and 85%, respectively; and for only DCP (>7.5 ng/mL) were 39%, 90%, 48%, and 86%, respectively. Values increased when AFP values were combined with AFP-L3% and DCP to 77%, 59%, 32%, and 91%, respectively. Among patients with increases in AFP levels to 20 to 200 ng/mL, AFP-L3% and DCP were highly specific markers (86.6% and 90.2%, respectively). Of 29 HCC patients with AFP levels less than 20 ng/mL, 13 had increased levels of AFP-L3% or DCP. Increased alanine aminotransferase levels were associated with increased total AFP but not AFP-L3% or DCP levels. Both AFP-L3%- and DCP-positive patients showed significant differences in lower cumulative HCC-free rates compared with the overall group (P < .0001 and P = .0005, respectively). CONCLUSIONS: AFP-L3% and DCP levels have higher correlation values with an absence of HCC, as well as a higher specificity and negative predictive value, than total AFP. Although this combination of markers only marginally improves surveillance for early HCC, it could identify individuals with negative imaging results who would benefit from follow-up evaluation.