Lectin Affinity Electrophoresis Research Articles

Rifampicin induces the bone form of alkaline phosphatase in humans.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic functions of PXR activation, we observed that rifampicin, a well-established ligand for human PXR, 600mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow-derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α-carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.

Development of on-chip fully automated immunoassay system "μTASWako i30" to measure the changes in glycosylation profiles of alpha-fetoprotein in patients with hepatocellular carcinoma.

Glycosylation profiles significantly change during oncogenesis. Aberrant glycosylation can be used as a cancer biomarker in clinical settings. Different glycoforms can be separately detected using lectin affinity electrophoresis and lectin array-based methods. However, most methodologies and procedures need experienced technique to perform the assays and expertise to interpret the results. To apply glycomarkers for clinical practice, a robust assay system with an easy-to-use workflow is required. Wako's μTASWako i30, a fully automated immunoanalyzer, was developed for in vitro diagnostics based on microfluidic technology. It utilizes the principles of liquid-phase binding assay, where immunoreactions are performed in a liquid phase, and electrokinetic analyte transport assay. Capillary electrophoresis on microfluidic chip has enabled the detection of different glycoform types of alpha-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma. AFP with altered glycosylation can be separated based on the reactivity to Lens culinaris agglutinin on electrophoresis. The glycoform AFP-L3 was reportedly more specific in hepatocellular carcinoma. This assay system can provide a high sensitivity and rapid results in 9 min. The test results for ratio of AFP-L3 to total AFP using μTASWako i30 are correlated with those of conventional methodology. The μTASWako assay system and the technology can be utilized for glycosylation analysis in the postgenomic era.

Lectin affinity electrophoresis.

An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

Analysis of Polarized Secretion of Fucosylated Alpha-Fetoprotein in HepG2 Cells

Fucosylated alpha-fetoprotein (AFP) is a more specific biomarker for hepatocellular carcinoma (HCC) than AFP. However, the mechanisms underlying the increase in fucosylated AFP in sera of HCC patients remain largely unknown. Recently, we reported that fucosylation is a possible signal for the secretion of hepatic glycoproteins into bile and that the fucosylation-based sorting machinery might be disrupted in the liver bearing HCC. In this study, we investigated the selective secretion of fucosylated AFP into bile canaliculus (BC) structures of the human hepatoma cell line HepG2. The proportion of fucosylated AFP in BC structures was higher than that in the medium, as judged by lectin affinity electrophoresis. Suppression of fucosylation by the double knock-down of GDP-mannose-4,6-dehydratase and the human homologue of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, which contribute to the synthesis of GDP-fucose, a donor substrate for fucosyltransferases, did not decrease the proportion of fucosylated AFP in BC structures but decreased this proportion in conditioned medium. Furthermore, increased AFP fucosylation was observed in medium, but not in BC structures, upon adding free fucose. These results suggest that saturation of fucosylated AFP in BC structures is accompanied by its increase in conditioned medium, probably leading to increased fucosylated AFP in sera of HCC patients.

Lectin affinity electrophoresis of serum alkaline phosphatase in metastasized breast cancer.

The use of serum alkaline phosphatase (ALP) isoenzymes as markers of breast cancer metastases and treatment efficacy has received little attention. Twenty-six breast cancer women (56+/-13 years, all post-menopausal) were prospectively evaluated during their first and third course of chemotherapy (4-week interval). Serum samples were analyzed for ALP isoenzymes (bone, liver, and intestine) using a lectin affinity electrophoresis kit (Hydragel 15 ISO-PAL, Sebia) adapted on a semi-automated Hydrasys system (Sebia). Results were compared with imaging techniques for the presence of metastases; bone ALP isoenzyme (B-ALP) results were compared with C-Terminal degradation products of type I collagen (S-CTX) (CrossLaps, IDS Nordic). Serum B-ALP, but not S-CTX, confirmed the presence of bone metastases (BM) (n=15) with 67/100% sensitivity/specificity (using a 69 UI/L ROC cut-off); ROC AUC was 0.806 (P=0.0004) (NS for S-CTX). Chemotherapy reduced serum B-ALP by 24% over 4 weeks (P=0.0012); there was no change for S-CTX. There was no specific clinical pattern for other ALP isoenzymes (liver and intestine). In conclusion, serum B-ALP, but not S-CTX, could help confirm the presence of BM in breast cancer patients.

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

Implementation of the on-chip immunoassay for α-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 10 min. 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 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/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.

Lens culinaris agglutinin-reactive α-fetoprotein and protein induced by vitamin K absence II are potential indicators of a poor prognosis: a histopathological study of surgically resected hepatocellular carcinoma

We histopathologically examined Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3)-positive hepatocellular carcinoma (HCC) and protein induced by vitamin K absence (PIVKA) II-positive HCC to clarify the efficacy of these markers for predicting a poor prognosis. Serum AFP-L3 and PIVKA II was measured in 110 HCC patients. AFP-L3 was measured by lectin-affinity electrophoresis coupled with antibody-affinity blotting, and PIVKA II by using a high-sensitivity kit. The growth type, capsule formation, capsule infiltration, portal vein invasion, intrahepatic metastasis and histological tumor grade were evaluated pathologically. Thirty-eight (35%) HCC patients were AFP-L3-positive, and 63 (57%) were PIVKA II-positive. In AFP-L3-positive HCC, the frequencies of an infiltrative growth type (positive : negative = 66% : 42%, P = 0.027) and a poorly differentiated type (positive : negative = 32% : 6%, P < 0.001) were significantly higher than in AFP-L3-negative HCC. In PIVKA II-positive HCC, the frequencies of an infiltrative growth type (positive : negative = 62% : 28%, P < 0.001), vascular invasion (positive : negative = 63% : 26%, P < 0.001), and intrahepatic metastasis (positive : negative = 38% : 4%, P < 0.001) were significantly higher than in PIVKA II-negative HCC. In both AFP-L3- and PIVKA II-positive HCC, the frequency of a poorly differentiated growth type was significantly higher than in HCC positive for either AFP-L3 or PIVKA II or HCC negative for both AFP-L3 and PIVKA II (both positive : either positive : both negative = 37% : 12% : 0%; P = 0.014, P < 0.001, respectively). AFP-L3 was related to progression from moderately differentiated to poorly differentiated HCC, whereas PIVKA II was more specific to vascular invasion. PIVKA II is therefore likely to be a useful indicator of vascular invasion.

AFP-L3 in Chronic Liver Diseases with Persistent Elevation of Alpha-fetoprotein

Alpha-fetoprotein (AFP) is an important marker for hepatocellular carcinoma (HCC). However, persistent elevation of AFP is found in patients with chronic liver diseases. The value of AFP-L3, which is more specific than AFP, was examined in such patients. We enrolled patients without image-detectable tumor, but with transient AFP value > 900 ng/mL (group A) or with persistent AFP value > 50 ng/mL for longer than 6 months (group B). Forty-one patients with HCC and AFP value > 50 ng/mL were included as the HCC control group (group C). AFP-L3 measurement was done by lectin-affinity electrophoresis coupled with antibody-affinity blotting. The study patients were followed with AFP, liver biochemistry and abdominal ultrasound at 3- to 6-month intervals. Additional studies were done when a tumor was suspected. One of 17 patients in group A and 13 of 39 patients in group B developed HCC within 2 years. When the cutoff value of AFP-L3 ratio was 15%, both the sensitivity and specificity were 71% for prediction of HCC during the next 2 years in all patients. Ninety percent of tumors larger than 5 cm had AFP-L3 > 15%, compared with only 60% for tumors smaller than 2 cm. Three patients in group A had AFP-L3 ratio > 17.5%. One patient developed HCC 10 months later; the other 2 patients were associated with hepatic failure. AFP-L3 provides a clue in HCC detection in patients with persistent elevation of AFP. However, AFP-L3 could be highly elevated in severe hepatitis.

Staging Hepatocellular Carcinoma by a Novel Scoring System (BALAD Score) Based on Serum Markers

Previously proposed staging systems for hepatocellular carcinoma (HCC) involve clinical, imaging, or pathologic factors in the evaluation. We established and validated a novel staging system for HCC that is based on simply serum markers. The new scoring system is based on 5 serum markers: bilirubin, albumin, Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3), alpha-fetoprotein (AFP), and des-gamma-carboxy prothrombin (DCP) and thus is termed the BALAD score. The system was validated in 2600 HCC patients from 5 institutions. The power of our system to predict patient survival and its discriminative ability were compared with those of previously reported staging systems. The best tumor marker cutoff values were 400 ng/dL for AFP, 15% for AFP-L3, and 100 milli-arbitrary unit/mL for DCP. The patients were classified into 6 categories on the basis of 5 laboratory values. The categories reflected patient survival well. The discriminative ability was comparable to that of previously reported staging systems. The new staging system for HCC combining serum albumin, serum bilirubin, and 3 tumor markers predicts patient outcomes with excellent discriminative ability. The system is easy to use and objective. In addition, stage can be evaluated with the use of only 1 serum sample. It also allows global comparison of patients with HCC or comparison of patients from different time periods with the same standard.

Further resolution of α-fetoprotein glycoforms by two-dimensional isoelectric focusing and lectin affinity electrophoresis

Human alpha-fetoprotein (AFP) from serum of patients with cirrhosis and hepatocellular carcinoma (HCC) was separated into several bands by IEF and by erythroagglutinating phytohemagglutinin (E-PHA) affinity electrophoresis. These AFP bands were directly compared in 2-D IEF and E-PHA affinity electrophoresis. IEF of serum AFP was run in 1% agarose IEF gel with 3% Pharmalyte 4.5-5.4. After IEF, a part of the gel was stained for AFP and another part of the gel corresponding to the area of separated AFP bands was cut in 1 mm x 39 mm along the focused direction and transferred to a trough in 1% agarose gel with 0.3 mg/mL E(4)-PHA for second-dimensional affinity electrophoresis. Separated 2-D AFP spots were visualized by antibody-affinity blotting and identified by combining the systems of Johnson et al.. (Johnson, P. J., Ho, S., Cheng, P., Chan, A. et al.., Cancer 1995, 75, 1663-1668) for AFP-I-+IV and of Taketa et al.. (Taketa, K., Ichikawa, E., Taga, H., Hirai, H., Electrophoresis 1985, 6, 492-497) for AFP-P1-5. AFP-P2, the major AFP glycoform, was composed of AFP-I, AFP+I, and AFP+II; AFP-P3, a nonspecific monosialo-AFP, was composed of AFP+II; AFP-P4, HCC-specific monosialo-AFP, was composed of AFP+II, AFP+III, and AFP+IV; and malignancy-related AFP-P5 was composed of AFP+I and AFP+II. Monosialo-AFP (AFP+II) was recovered in all the glycoforms of AFP-P2, -P3, -P4, and -P5; thus, AFP-P4 is more specific to HCC than monosialylated AFP+II.

Lectin‐reactive α‐Fetoprotein in Patients with Tyrosinemia Type I and Hepatocellular Carcinoma

Despite the introduction of 2-(2-nitro-4-trifluormethyl-benzoyl)-1,3-cyclohexandion into the treatment of hereditary tyrosinemia type I (HT1), patients remain at risk of developing hepatocellular carcinoma (HCC). Serial total alpha-fetoprotein (AFP) levels are used to monitor the individual patient. Lectin-reactive alpha-fetoprotein (L3-AFP) is an AFP isoform that is expressed by malignant liver tumors. We investigated whether the analysis of L3-AFP could lead to earlier detection of HCC in HT1 compared with judgement based on total AFP alone. AFP electrophoresis using lectin-containing agarose gel identifies L3-AFP by the affinity of its specific carbohydrate chain to lectin. We report the retrospective analysis of sequential serum samples of 12 patients with HT1 and histologically proven HCC. AFP isoforms could be identified in all 12 patients. In 6 patients, the L3-AFP increased before the total AFP. In 3 patients, the rise in L3-AFP was parallel to the rise of total AFP; and in 3 patients, the L3-AFP was raised after the total AFP or did not increase at all. We were able to identify 6 of 12 patients with an early increase in the new tumor marker. Lectin-affinity electrophoresis may have a role in discriminating benign liver disease from HCC in HT1. We suggest the further evaluation of L3-AFP in HT1.

Prognostic Significance of Simultaneous Measurement of Three Tumor Markers in Patients With Hepatocellular Carcinoma

We conducted a prospective study to evaluate the significance of simultaneous measurement of 3 currently used tumor markers in the evaluation of tumor progression and prognosis of patients with hepatocellular carcinoma (HCC). Three tumor markers for HCC, alpha-fetoprotein (AFP), Lens culinaris agglutinin A-reactive fraction of AFP (AFP-L3), and des-gamma-carboxy prothrombin (DCP), were measured in the same serum samples obtained from 685 patients at the time of initial diagnosis of HCC. Positivity for AFP >20 ng/dL, AFP-L3 >10% of total AFP, and/or DCP >40 mAU/mL was determined. In addition, tumor markers were measured after treatment of HCC. Of the 685 patients, 337 (55.8%) were positive for AFP, 206 (34.1%) were positive for AFP-L3, and 371 (54.2%) were positive for DCP. In a comparison of patients positive for only 1 tumor marker, patients positive for AFP-L3 alone had a greater number of tumors, whereas patients positive for DCP alone had larger tumors and a higher prevalence of portal vein invasion. When patients were compared according to the number of tumor markers present, the number of markers present clearly reflected the extent of HCC and patient outcomes. The number of markers present significantly decreased after treatment. Tumor markers AFP-L3 and DCP appear to represent different features of tumor progression in patients with HCC. The number of tumor markers present could be useful for the evaluation of tumor progression, prediction of patient outcome, and treatment efficacy.

A study of oligosaccharide variants of alpha‐fetoproteins produced by normal fetuses and fetuses with trisomy 21

The mechanisms of the increase in the percentage of alpha-fetoproteins (AFPs) that strongly binds to Lens culinaris agglutinin (AFP-L3) in pregnancies with a trisomy 21 fetus have not been analyzed. To investigate the oligosaccharide variants of AFP produced by normal fetuses and fetuses with trisomy 21, the lectin reactivity of AFP was analyzed. Fetal liver tissue, amniotic fluid, and maternal serum were obtained from five normal pregnancies and five pregnancies with a trisomy 21 fetus. The percentages of AFP reactive to lectins were determined by lectin-affinity electrophoresis coupled with antibody-affinity blotting. The percentages of AFP-L3 in the fetal liver and the maternal serum were 23.9 and 27.0%, respectively, in normal pregnancies, and 28.7 and 38.5%, respectively, in pregnancies with a trisomy 21 fetus. There was no statistically significant difference between the percentage in the fetal liver and the percentage in the maternal serum in normal pregnancies; however, a significant difference (P < 0.01) was found in pregnancies with a trisomy 21 fetus. In regard to the percentage of AFP-L3 in the fetal liver, there was no significant difference; however, a significant difference (P < 0.05) was found in the maternal serum between normal pregnancies and pregnancies with a trisomy 21 fetus. The transference of the AFP-L3 fraction might be relatively high in the placentas of women carrying a trisomy 21 fetus, and this could be the one of the reasons for the increase in the percentage of AFP-L3 in the maternal serum in pregnancies with a trisomy 21 fetus.

Relationship between Lens culinaris agglutinin‐reactive α‐fetoprotein and pathologic features of hepatocellular carcinoma

We investigated pathological features of Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3)-positive hepatocellular carcinoma (HCC) in order to seek a pathological basis of poor prognosis of HCC patients with elevated AFP-L3. A total of 111 patients with HCC < or =5 cm in diameter who underwent hepatic resection were studied. Serum AFP-L3 concentration was measured within a month prior to surgery by lectin-affinity electrophoresis coupled with antibody-affinity blotting, and expressed as AFP-L3 percentage of total AFP. AFP-L3 of 10% or higher was judged to be positive. Pathologic features of resected HCC specimens were evaluated and classified concerning growth pattern (expansive or infiltrative growth), capsule formation, capsule infiltration, septal formation, portal vein invasion, hepatic vein invasion, bile duct invasion, and intrahepatic metastasis. These macroscopic and microscopic findings were compared between AFP-L3-positive and negative HCC specimens. Thirty-three (29.7%) were positive for AFP-L3. The prevalence of HCC with infiltrative growth, with capsule infiltration, with septum formation, with portal vein invasion, and with hepatic vein invasion was significantly higher in AFP-L3-positive group (P=0.0121, 0.0290, 0.0442, 0.0314, and 0.0433, respectively). These pathologic features reportedly indicate the progression of the tumor. AFP-L3-positive HCC had several pathologic features of progressed state of HCC, which accounted for the AFP-L3 as an indicator of poor prognosis of HCC.

Lektin-reaktives Alpha-Fetoprotein bei Patienten mit Tyrosinämie Typ I

Despite the introduction of NTBC into the treatment of tyrosinaemia type I (TT1) and a considerable improvement in the outcome of these patients, the principal risk of developing hepatocellular carcinoma (HCC) in this metabolic disorder remains mainly in those children with late introduction of NBTC after the second year of life. Serial total alpha-Fetoprotein (AFP) levels are used to evaluate the individual risk to develop malignant changes. A failure of AFP to decrease on adaequate treatment or a secondary increase after a period of falling levels have been an indication for liver transplantation. Lectin-reactive alpha-Fetoprotein is a recently described marker to distinguish hepatocellular carcinoma from benign liver disease in adult cirrhotic patients. To investigate if the analysis for Lectin-reactive alpha-Fetoprotein would lead to earlier detection of HCC compared to a judgement based on the evolution of standard total AFP alone. We report the analysis of 12 patients with TTI and histologically proven HCC. There of 5 were diagnosed under one year of age, but NTBC treatment was started between 2 years 3 month and 7 years of age except in one case in which NTBC was introduced when the diagnosis of TTI was made. The remainder of the patients cover up to the age of 15 years. All patients had been treated with NTBC. Lectin containing agarose gel for AFP electrophoresis leads to AFP separation according to different affinities of the varying carbohydrate chains of AFP to lectins. AFP subfractions could be identified in all 12 patients. In 6 patients the L3-AFP rose before the total AFP. In 3 patients the rise in L3-AFP was consistent with the rise of the total AFP and in 3 patients the L3-AFP was raised after the total AFP or did not increase at all. We were able to identify 6 out of 12 patients who had an early increase of L3-AFP before they developed a change in total AFP levels. The clinical significance of these early changes need to be determined. Lectin-affinity electrophoresis may have a potential role as an additional tool that may help to discriminate benign liver disease from HCC in TTI. We suggest the further evaluation of lectin-reactive AFP in TTI.

Changes of Serum Alkaline Phosphatase Activity in Dry and Lactational Cows

Serum alkaline phosphatase (ALP) activities were measured during dry and lactational periods to investigate the influence of lactation on serum ALP activity in cows. Higher levels of serum ALP activity were seen in lactational periods than in dry periods. The serum activities of bone-specific ALP (BALP), liver ALP (LALP), tartrate resistant acid phosphatase (TRAP) and aspartate aminotransferase also increased in lactational periods. ALP activities in the bone extract and in whey were decreased at similar rates by the addition of lectin. Moreover, since the ALP band in whey was observed to have the same migration in polyacrylamide gel (PAG) disk electrophoresis as that of the bone extract, analysis of ALP isoenzymes by lectin affinity or PAG disk electrophoresis could not distinguish ALP originating from the mammary gland from that of bone. In this study, it was clear that the increased level of serum ALP activity was due to increases of BALP and LALP in lactational periods. However, the extent of the influence of ALP originating from the mammary glands on serum ALP activity was unknown. Judging from changes of BALP and TRAP activities in the serum and the correlation between the both, it was guessed that ALP originating from the mammary glands influenced serum ALP activity.

A study on the microheterogeneity of alpha-fetoproteins produced by yolk sac and germ cell tumors.

It is generally believed that the lower the grade of differentiation of glycoprotein-producing cells, the more often modification by bisecting N-acetylglucosamine (GlcNAc) or fucose (Fuc) at the sugar chain of the glycoprotein or increase in branching of side chains occurs. We examined the characteristics of the alpha-fetoprotein (AFP) sugar chain stored in amniotic and exocoelomic fluid during 5-9 weeks of gestation and analyzed serum-derived AFP of patients with germ cell tumors. Total AFP concentrations in embryonic fluid at 5-9 weeks of gestation (n = 11) and serum of patients with germ cell tumors (n = 7) were measured using a radioimmunoassay (RIA) method. The percentages of AFPs reactive with Lens culinaris agglutinin (LCA), concanavalin A (Con A), erythroagglutinating phytohemagglutinin-E4 (E-PHA) and Ricinus communis agglutinin-120 (RCA 120) were obtained by lectin-affinity electrophoresis coupled with antibody-affinity blotting. It was revealed that at 5-9 weeks of gestation, AFP variants that had been modified by the Fuc residue, which bound to the GlcNAc residue at the reducing end of the sugar chain, and bisecting GlcNAc residues gradually decreased as pregnancy advanced; however, the presence of N-acetylneuraminic acid (Neu5Ac) at the nonreducing ends changed little. It appears very likely that the changes in the relative amounts of AFP variants in the embryonic fluid during early pregnancy were due to differentiation of the yolk sac. The grade of differentiation of yolk sac tumors was very similar to that of the normal yolk sac at around 6 weeks of gestation.

Lens culinaris agglutinin-reactive alpha-fetoprotein as a prognostic marker in patients with hepatocellular carcinoma undergoing transcatheter arterial chemoembolization.

Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3) is known to be a useful marker for the diagnosis of hepatocellular carcinoma (HCC). Recent studies have shown that positive AFP-L3 results after treatment predicts tumor recurrence and poor clinical outcome. This study was to evaluate the role of pretreatment AFP-L3 as a prognostic marker for response to transcatheter arterial chemoembolization (TACE) and survival in patients with HCC. Forty-six patients with HCC who underwent TACE were analyzed. Agglutinin-reactive AFP was measured by lectin-affinity electrophoresis coupled with antibody-affinity blotting. Agglutinin-reactive AFP results larger than 24.4% were considered to be positive. Agglutinin-reactive AFP fractions were positive in 32 patients. Agglutinin-reactive AFP-positive patients had poorer performance status, larger tumors, frequent portal vein thrombosis, and higher levels of serum AFP. The partial response rate to TACE was lower in AFP-L3-positive patients than in AFP-L3-negative ones (37.5% vs. 78.6%, p = 0.01). Tumor size and AFP-L3 were two independent predictive factors for response to TACE. The 2-year survival rate was lower in AFP-L3-positive patients than in AFP-L3-negative ones (21.2% vs. 78.6%, p = 0.01). Child-Pugh class, AFP-L3, the presence of portal vein thrombosis, and response to TACE were independent prognostic factors for survival. In conclusion, pretreatment status of AFP-L3 could be considered a useful marker for predicting clinical outcome in patients with HCC who underwent TACE.

Simultaneous determination of percentage of Lens culinaris agglutinin-reactive α-fetoprotein and α-fetoprotein concentration using the LiBASys clinical auto-analyzer

Background: Lens culinaris agglutinin (LCA)-reactive α-fetoprotein (AFP-L3) percentage of total AFP concentration [(AFP-L3/total AFP)×100] has been used as an effective marker for earlier diagnosis, for assessment of therapeutic effects and for predicting the prognosis of hepatocellular carcinoma (HCC). Methods: A new clinical automatic analyzer, the “LiBASys”, and an assay kit for the simultaneous determination of AFP-L3 percentage and concentration of AFP in human serum were developed. LiBASys performed automatic re-measurement and also met stat samples. Both AFP-L3 percentage and AFP concentration were calculated simultaneously and printed out continuously every 3.4 min per test. Results: A linear dose–response relationship was observed up to 1000 ng/ml AFP concentration. The detection limit and functional sensitivity of LCA-nonreactive AFP (AFP-L1) and AFP-L3 concentrations were 0.4 and 0.8 ng/ml, and 0.4 and 1.0 ng/ml, respectively. Interassay CVs of AFP-L3 percentage and AFP concentration ranged from 2.8% to 13.4% and 2.6% to 4.6%, respectively. Assay results exhibited good correlations with those of commercially available lectin-affinity electrophoresis ( r=0.984) for AFP-L3 percentage and with the RIA method ( r=0.999) for AFP concentration. Conclusions: This method simultaneously yields both qualitative and quantitative results for AFP-L3 percentage and AFP concentration.

Lens culinaris agglutinin-reactive α-fetoprotein, an alternative variant to α-fetoprotein in prenatal screening for Down's syndrome

Three serum tests, alpha-fetoprotein (AFP), human chorionic gonadotrophin and unconjugated oestriol, are now widely used for screening for Down's syndrome. Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3) is a variant of alpha-fetoprotein with alpha1-->6 fucose appended to the reducing terminal N-acetylglucosamine. It is the most prominent AFP detected in the serum of patients with hepatocellular carcinoma. We investigated microheterogeneities of the carbohydrate chain on AFP in fetal liver tissues, amniotic fluids and maternal sera obtained from pregnancies with Down's syndrome using lectin affinity electrophoresis with four lectins. The percentages of AFP-L3 in maternal sera from 22 Down's syndrome and 227 unaffected pregnancies were determined. Unlike the case with AFP concentration, the percentage of AFP-L3 in maternal serum and amniotic fluid was similar, and apparently not influenced by membrane permeability. Knowing the percentage of AFP-L3 in maternal serum was effective for discriminating between Down's syndrome-affected pregnancies and unaffected pregnancies. The percentage of AFP-L3 in maternal serum identified 55% of Down's syndrome cases with a 5% false-positive rate. AFP-L3 should be an effective replacement for AFP in prenatal Down's syndrome screening.