Development of Des-_-carboxy Prothrombin

Clin Chem Lab Med 2008;46(3):411416 2008 by Walter de Gruyter Berlin New York. DOI 10.1515/CCLM.2008.078 2007/424Article in press - uncorrected proof

Development of des-g-carboxy prothrombin (DCP) measuringreagent using the LiBASys clinical analyzer

Isao Yamaguchi*, Kenji Nakamura, HiromichiKitano, Yoshie Masuda, Futoshi Kanke, ShinzoKobatake and Shinji Satomura

New Diagnostics Business and TechnologyDevelopment Department, Wako Pure ChemicalIndustries Ltd., Hyogo, Japan

Abstract

Background: Des-g-carboxy prothrombin (DCP), Lensculinaris agglutinin-reactive a-fetoprotein ratio (AFP-L3) and total a-fetoprotein (AFP) are tumor markersuseful for diagnosing and determining the prognosisof hepatocellular carcinoma (HCC). There is a realneed for measurement of these three markers on aone-assay platform.Methods: A method of DCP measurement in humanserum was developed using liquid binding assay(LBA), which enables rapid antigen-antibody reactionand bound/free separation on the LiBASys clinicalanalyzer.Results: The dilution curve for DCP was linear up to500 ng/mL. The limit of detection of DCP concen-tration was 0.5 ng/mL. Intra- and inter-assay coeffi-cients of variation of DCP were 0.7%2.4% and2.2%6.5%, respectively. This method was free frominterference by hemoglobin, bilirubin, ditaurobiliru-bin, intrafat, ascorbate, galactose, glucose and rheu-matoid factor. The analytical recoveries of DCP addedto serum were 91.7%108.2%. DCP concentrationmeasured with the LBA method was linear and wassignificantly correlated with that measured with theELISA method.Conclusions: The LiBASys clinical analyzer madepossible measurement of the complementary tumormarkers, HCC, total AFP, AFP-L3 and DCP.Clin Chem Lab Med 2008;46:4116.

Keywords: des-g-carboxy-prothrombin; hepatocellu-lar carcinoma; Lens culinaris agglutinin-reactive a-fetoprotein ratio (AFP-L3); LiBASys; total a-fetoprotein(AFP).

Introduction

Prothrombin is a vitamin K-dependent coagulationfactor that is activated to thrombin during blood clot-

*Corresponding author: Isao Yamaguchi, New DiagnosticsBusiness and Technology Development Department, WakoPure Chemical Industries, Ltd., 6-1 Takada-cho, Amagasaki,Hyogo, 661-0963, JapanPhone: q81-6-6499-9109, Fax: q81-6-6499-1524,E-mail: [email protected] September 10, 2007; accepted November 23, 2007;previously published online February 6, 2008

ting (1). It is formed in the liver, when the 10 glutamicacid (Glu) residues in the amino terminal in its pre-cursor are converted to g-carboxyglutamic acid (Gla)by a vitamin K-dependent carboxylase in the post-translational process (2). When all or some of the 10Glu residues are not converted to Gla, an immatureform of prothrombin is secreted into the blood,termed des-g-carboxy prothrombin (DCP) or proteininduced by vitamin K absence or antagonist-II (PIVKA-II) (3).

Liebman et al. showed that DCP is a serum tumormarker for primary hepatocellular carcinoma (HCC)(4). DCP has since been investigated and used as aspecific tumor marker for HCC in Japan. a-Fetoprotein(AFP) is an oncofetal glycoprotein that has been usedas a tumor marker for HCC. However, AFP is not spe-cific to HCC. Benign liver diseases, such as chronichepatitis and cirrhosis, are known to increase AFPconcentration in blood. Recently, the Lens culinarisagglutinin-reactive a-fetoprotein ratio (AFP-L3), whichhas an a16 fucose residue appended to N-acetylglu-cosamine at the reducing end, has been reported tobe a specific marker for HCC. In Japan, DCP and AFP-L3%, the ratio of AFP-L3 to total AFP, have been wide-ly and routinely used as serum markers for HCC. Ithas been reported that the rate of detection of smallHCC is improved by a combination assay with DCPand AFP-L3 and that these markers are complemen-tary and useful for the diagnosis and evaluation ofsmall HCC when measured simultaneously (5, 6).

We developed the LiBASys clinical analyzer basedon the liquid binding assay (LBA) method (7, 8). In theLBA method, the antigen-antibody reaction is formedin the liquid phase to enable optimization of the anti-body concentration and greatly shortens antigen-anti-body reaction times. We have already establishedsimultaneous determination of both total AFP andAFP-L3 with the LiBASys (8). As the combination ofDCP and AFP-L3% measurements improves HCCdetection, a DCP assay was developed on the LiBASysinstrument, making single-platform measurement ofDCP and AFP-L3% possible.

Materials and methods

Materials

All serum samples were stored below y808C until measure-ment. Sulfated tyrosine-pentamer (YS5) was synthesized andwas labeled with the single binding moiety (Fab9) of anti-prothrombin monoclonal antibody (mouse) to prepare Fab9-YS5 antibody. Peroxidase (POD) was from Toyobo (Osaka,Japan) and was labeled with the Fab9 of anti-DCP monoclo-nal antibody (mouse) to prepare Fab9-POD. Anti-DCP mono-clonal antibody is a highly specific antibody to DCP prepared

412 Yamaguchi et al.: Development of DCP measuring reagent using the LiBASys

Article in press - uncorrected proof

using a synthetic peptide (1135 positions) as the antigenand the cell line SP2/O as a myeloma. Serum containinghuman anti-mouse antibody (HAMA) was from Roche (Basel,Switzerland). DCP concentration was measured using anenzyme-linked immunosorbent assay method (Eitest PIVKA-II, Eisai, Tokyo, Japan). AFP-L3 and total AFP were measuredusing the LBA method (LBA AFP-L3, Wako, Osaka, Japan).Purified DCP was prepared using a thermal decarboxylationprocedure and by using affinity chromatography with anti-DCP monoclonal antibody.

Determination of DCP concentration

The purified DCP was isolated from decarboxylated pro-thrombin by heating and was purified by immuno-affinitychromatography with a monoclonal antibody specific forDCP (clone PI-1). Clone PI-1 does not recognize normal pro-thrombin. The affinity column was prepared using NHS-acti-vated Sepharose 4 Fast Flow Lab Packs (GE Healthcare, LittleChalfont, UK). DCP contents of the column eluates in thepurification steps were monitored by determining absor-bance at 280 nm. The purified DCP was tested for a singleprotein band on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). The protein concentration ofthe purified DCP (ng/mL) was determined using the BCA pro-tein assay kit (Pierce, Rockford, IL, USA) according to themanufacturers protocols. The DCP concentration of purifiedDCP wmilli-arbitrary unit (mAU)/mLx was determined usingan Eitest PIVKA-II. We converted from the conventional unit(AU) to the SI unit (g) and calculated the DCP conversionfactor from measurement data.

Apparatus

Automated assay of DCP concentration was performed withthe LiBASys clinical analyzer (liquid binding assay system;Wako, Japan). The LiBASys consists of three compartments:outer and inner cuvettes for immune and enzyme reactions,an anion-exchange column for bound/free (B/F) separationand an optical assembly for fluorophotometric detection.With the LiBASys, the antigen-antibody reaction is per-formed in the liquid phase, and B/F separation is rapidlyperformed using column chromatography without a solidphase. The outer cuvettes are incubated at 378C. The innercuvettes and sample carousel are cooled below 108C.

Reagents

The reagent consisted of Reagent, Substrate 1 and Substrate2. The Reagent consists of 39.7 nmol/L Fab9-POD and235 nmol/L Fab9-YS5 in 50 mmol/L N-(2-acetamido)-2-aminoethanesulfonate buffer, pH 6.5. Opened Reagent canbe used for 10 days at 2108C on the LiBASys. Substrate 1consists of 320 mmol/L 4-acetoamidophenol in 2-propanol.Substrate 2 consists of 40 mmol/L hydrogen peroxide in15 mmol/L citrate buffer, pH 5.5. One bottle of Substrate 1 isadded to one bottle of Substrate 2, mixed well, and used asthe working substrate solution. Mixed substrate solution isstable for 10 days when stored at 2108C. Wash solution con-sists of 1.0 mol/L citrate. Elution buffers A, B and C consistof 0.27, 0.90 and 3.0 mol/L NaCl in 50 mmol/L Tris-HCl buffer,pH 8.0. The proposed standard of DCP for the assay is pre-pared with human prothrombin decarboxylated by heating.(9).

Assay procedure

A total of 30 mL of sample containing DCP and 100 mL ofReagent are mixed in inner cuvettes and reacted at 108C for

5 min. Fab9-POD and Fab9-YS5 bind to DCP and form immunecomplexes. After incubation, 80 mL of the reaction mixtureis introduced into an anion-exchange column. The immunecomplexes have affinity for the column, but none for freeFab9-POD. The column is then washed with Elution buffer A,free Fab9-POD, and the sample is eluted from the column.Finally, immune complex fractions are eluted from thecolumn with Elution buffer C. A total of 900 mL of eluate ispoured into outer cuvettes incubated at 378C. In addition,100 mL of substrate solution is added to eluate containingimmune complexes, and the POD activity of the immunecomplexes is then measured. POD activity is determined asthe increase in fluorescence intensity. These values are com-pared to fluorescence intensities of known standards for DCPconcentration to obtain the DCP concentrations of samples.

Linearity

The dilution curve was prepared by plotting the measuredDCP concentration on the y-axis vs. the dilution ratio of DCPon the x-axis. A high DCP value sample was prepared byspiking DCP to a pool serum and diluted. Saline was usedfor sample dilution.

Limit of detection

The minimum limit of detection was determined in eight rep-licates of samples containing known amounts of DCP. TheDCP concentration that corresponded to the zero standardplus two times the standard deviation (SD) was determinedto be the limit of detection.

Precision

Serum samples were used to determine the intra- and inter-assay coefficients of variation (CVs). In the intra-assay test,sample was assayed in 21 replicates, while in the inter-assaytest, the same sample was assayed in 21 replicates on dif-ferent days for 28 days. Intra- and inter-assay precisions oneach LiBASys were also examined.

Recovery

The recovery of DCP from serum samples was measured byadding known amounts of DCP to serum samples.

Interference studies

Several endogenous substances, including hemoglobin, bil-irubin, ditaurobilirubin, intrafat, ascorbate, galactose, glu-cose, rheumatoid factor and HAMA were added to serumsamples to assess interference. Serum samples containingDCP without interference substances were measured as acontrol. Recovery of DCP using HAMA was examined toassess interference by adding known amounts of DCP toHAMA or normal serum.

Comparison of methods

Serum samples were measured with the LBA method andthe ELISA method. The DCP concentrations measured withthe LBA method were compared with those measured withthe ELISA method.

Yamaguchi et al.: Development of DCP measuring reagent using the LiBASys 413


Table 1 Results of determination of DCP conversion factor.

DCP lot SD Mean

A B C D

DCP, mAU/mL 4,723,000 4,950,000 3,814,000 4,000,000Protein, ng/mL 92,000 111,600 64,100 64,500DCP conversion factor, ng/mAU 0.019 0.023 0.017 0.016 0.003 0.019

Figure 1 SDS-PAGE of purified DCP samples.SDS-polyacrylamide slab gel electrophoresis was performedby the method of Laemmli with 7.5% running gel. Gels werestained with Coomassie Brilliant Blue R-250 for proteins.

Results

Determination of DCP concentration

Four lots of purified DCP samples were preparedusing a thermal decarboxylation procedure and puri-fied by using immune affinity chromatography withanti-DCP monoclonal antibody. Each sample was con-firmed using SDS-PAGE. SDS-PAGE was performedby the method of Laemmli (10) with 7.5% running gel.Gel was stained with Coomassie Brilliant Blue R-250for proteins. On SDS-PAGE, every sample migrated asa single band, indicating pure DCP (Figure 1).

DCP and protein concentrations of sample weredetermined using the BCA protein assay kit (ng/mL)and the DCP detection kit (mAU/mL). The DCP con-version factor to convert from the conventional unit(AU) to the SI unit (g) for each sample was calculatedfrom measurement data. The DCP conversion factorsfor mAU to ng were 0.016 to 0.023 ng/mAU. Themeanvalue of samples was 0.019 ng/mAU, with a SD of0.003. For the reasons indicated above, the DCPconversion factor of mAU to ng was fixed at 0.019ng/mAU (Table 1).

Linearity

Typical dilution curves for the presented DCP assayare shown in Figure 2AC. Figure 2A is the dilutioncurve for high concentration, Figure 2B for inter-

mediate concentration and Figure 2C for lowconcentration.

Limit of detection

The dilution curve was linear between 0 and 500ng/mL. The analytical limit of detection of DCP con-centration was estimated as the dose equivalent tothe mean of eight replicates of the zero standard plus2 SD. The DCP concentration that corresponded tothis signal was calculated to be 0.50 ng/mL (Figure 3).

Precision, recovery and interference studies

The intra-assay (21 replicates) and inter-assay (21replicates for 28 days) CVs of DCP concentration were0.7%2.4% and 2.2%6.5%, respectively (Table 2).The analytical recovery of DCP added to serumranged from 91.7% to 108.2% (Table 3). Endogenoussubstances, including hemoglobin (02 g/L), bilirubin(04 mg/L), ditaurobilirubin (02 mg/L), intrafat(0%2%), ascorbate (05 mg/L), galactose (020mg/L), glucose (0100 mg/L) and rheumatoid factor(0550 IU/mL), did not interfere with the assay results.Recoveries of DCP added to HAMA serum rangedfrom 87.1% to 109.1%. HAMA did not interfere withassay results.

Comparison of methods

DCP concentrations measured by the LBA methodwere linear and were significantly correlated withthose measured by the ELISA method. The regressionformula for DCP was ys0.0194xy0.0468 (Figure 4;ysDCP concentration by LBA method, ng/mL; xsDCPconcentration by ELISA method, mAU/mL; ns49;rs0.9956).

Discussion

HCC is one of the most common cancers in the world.Most HCCs develop in cirrhotic liver, and patients withcirrhosis comprise a high-risk group for the develop-ment of HCC (11). Total AFP, AFP-L3 and DCP havebeen widely used clinically as tumor markers of HCCin Japan (4, 1214). Total AFP is a commonly usedtumor marker of HCC, exhibits acceptable sensitivityand generally reflects the amount of tumor mass (6).However, AFP sometimes increases not only in HCCpatients but also in cirrhosis patients (15, 16), anddoes not increase in 35%45% of HCC patients (17,18). It is thus difficult to distinguish early-stage HCC



Figure 2 Linearity.Dilution curves were prepared by plotting the DCP concentration on the y-axis vs. the mixing ratio on the x-axis. The dilutioncurve was linear between 0 and 500 ng/mL. (A) High concentration of DCP, (B) intermediate concentration of DCP and (C) lowconcentration of DCP.

Figure 3 Limit of detection.The limit of detection was determined from the mean con-centration of eight replicates of the zero standard plus 2 SD.The detection limit of DCP concentration is 0.50 ng/mL.

Table 2 Precision of DCP.

Sample Mean CV (%)DCP, ng/mL

Intra-assay Inter-assay

1 1.0 1.9 6.52 2.4 1.6 3.33 8.2 2.1 2.24 57.4 0.7 2.55 190.6 1.8 2.46 453.2 2.4 2.5

Values are given as means across all LiBASys analyzers.

patients from cirrhosis patients using total AFP alone.AFP-L3 is the ratio of the structure of carbohydratechain changes according to the cancerization of cellsand is the percentage of AFP with affinity for Lens

culinaris agglutinin against total AFP. It has beenreported that AFP-L3 is highly specific for HCC andyields positive results earlier than diagnosis of HCCby imaging modalities (12, 15, 19). In addition, it hasbeen reported that AFP-L3 is useful in determining theprognosis after treatment of HCC, as well as the bio-logical malignancy of HCC (20, 21). DCP is an imma-ture form of prothrombin, where all or some of the 10Glu residues are not converted to Gla. DCP showedstrong correlation with tumor size of HCC and can beused to detect early-stage HCC (2226). Izuno et al.reported a correlation between DCP level and theexistence of multiple HCCs (27). Suzuki et al. sug-gested that DCP concentration was a useful predictorof portal venous invasion in patients with HCC. It hasalso been reported that it is possible that DCP acts asan autologous mitogen and stimulates the prolifera-tion of HCC (28).

Because no correlations have been found betweenDCP and total AFP (2931) or between DCP and AFP-L3 (5, 6) in HCC patients, the combination assay withAFP-L3 and DCP was used to measure complemen-tary markers. It has been reported that a combinationassay improves the sensitivity in detection of HCC (5,6, 21, 30) and is useful for follow-up of cirrhosispatients (6). Toyoda et al. suggested that simultan-eous measurement of these tumor markers could beuseful for the evaluation of tumor progression, pre-diction of patient outcome and determination of treat-ment efficacy (32).

However, no clinical analyzer measuring both AFP-L3 and DCP has been available. We have alreadydeveloped a simultaneous determination reagent fortotal AFP and AFP-L3 with the LiBASys clinical ana-lyzer (8). In addition, a reagent for determination ofDCP concentration in serum on the LiBASys has beenestablished with excellent performance. Using theLiBASys, the combination assay of AFP-L3 and DCPwas easily performed. The LiBASys could be usefulfor the early detection and determination of the prog-nosis of HCC.

Yamaguchi et al.: Development of DCP measuring reagent using the LiBASys 415


Table 3 Recovery of DCP.

Sample DCP, DCP added, DCP measured, Obtained, Recovery, %ng/mL ng/mL ng/mL ng/mL

1 2.1 1.2 3.2 1.1 91.72.3 4.4 2.3 100.05.2 7.3 5.2 100.08.7 11.2 9.1 104.6

2 14.9 1.4 16.3 1.4 100.04.7 19.7 4.8 102.19.6 24.7 9.9 103.1

18.4 34.7 19.8 107.636.7 54.6 39.7 108.2

3 108.2 83.1 171.2 88.1 106.0193.2 387.3 194.1 100.5346.3 713.9 367.6 106.2

Figure 4 Comparison of DCP concentrations determined bythe LBA method and the ELISA method.DCP concentrations measured by the LBA method were sig-nificantly correlated with those measured by the ELISAmethod. The ELISA method (Eitest PIVKA-II, Eisai, Tokyo,Japan) allows measurement of DCP in the range of102000 mAU/mL.

References

1. Barton PG, Jackson CM, Hanahan DJ. Relationshipbetween factor V and activated factor X in the generationof prothrombinase. Nature 1967;27:9234.

2. Larson AE, Suttie JW. Vitamin K-dependent carboxylase:evidence for a hydroperoxide intermediate in the reaction.Proc Natl Acad Sci USA 1978;75:54136.

3. Suttie JW. Recent advances in hepatic vitamin K meta-bolism and function. Hepatology 1987;7:36776.

4. Liebman HA, Furie BC, Tong MJ, Blanchard RA, Lo KJ,Lee SD, et al. Des-gamma-carboxy (abnormal) prothrom-bin as a serum marker of primary hepatocellular carci-noma. N Engl J Med 1984;310:142731.

5. Sassa T, Kumada T, Nakano S, Uematsu T. Clinical utilityof simultaneous measurement of serum high-sensitivitydes-gamma-carboxy prothrombin and Lens culinarisagglutinin A-reactive alpha-fetoprotein in patients with

small hepatocellular carcinoma. Eur J GastroenterolHepatol 1999;11:138792.

6. Shimauchi Y, Tanaka M, Kuromatsu R, Ogata R, TateishiY, Itano S, et al. A simultaneous monitoring of Lensculinaris agglutinin A-reactive alpha-fetoprotein anddes-gamma-carboxy prothrombin as an early diagnosisof hepatocellular carcinoma in the follow-up of cirrhoticpatients. Oncol Rep 2000;7:24956.

7. Nakamura K, Satomura S, Matsuura S. Liquid-phasebinding assay of human chorionic gonadotropin usinghigh-performance liquid chromatography. Anal Chem1993;65:6136.

8. Yamagata Y, Shimizu K, Nakamura K, Henmi F, Sato-mura S, Matsuura S, et al. Simultaneous determinationof percentage of Lens culinaris agglutinin-reactivealpha-fetoprotein and alpha-fetoprotein concentration usingthe LiBASys clinical auto-analyzer. Clin Chim Acta 2003;327:5967.

9. Bajaj SP, Price PA, Russell WA. Decarboxylation ofgamma-carboxyglutamic acid residues in human pro-thrombin. J Biol Chem 1982;257:372631.

10. Laemmli UK. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature1970;227:6805.

11. Okuda K. Early recognition of hepatocellular carcinoma.Hepatology 1986;6:72938.

12. Abelev GI. Production of embryonal serum alpha-glob-ulin by hepatomas: review of experimental and clinicaldata. Cancer Res 1968;28:134450.

13. Taketa K, Endo Y, Sekiya C, Tanikawa K, Koji T, Taga H,et al. A collaborative study for the evaluation of lectin-reactive alpha-fetoproteins in early detection of hepato-cellular carcinoma. Cancer Res 1993;53:541923.

14. Yamashita F, Tanaka M, Satomura S, Tanikawa K. Prog-nostic significance of Lens culinaris agglutinin A-reactivealpha-fetoprotein in small hepatocellular carcinomas.Gastroenterology 1996;111:9961001.

15. Alpert E, Feller ER. Alpha-fetoprotein (AFP) in benign liv-er disease. Evidence that normal liver regeneration doesnot induce AFP synthesis. Gastroenterology 1978;74:8568.

16. Sato Y, Nakata K, Kato Y, Shima M, Ishii N, Koji T, et al.Early recognition of hepatocellular carcinoma based onaltered profiles of alpha-fetoprotein. N Engl J Med 1993;328:18026.

17. Peng YC, Chan CS, Chen GH. The effectiveness of serumalpha-fetoprotein level in anti-HCV positive patients forscreening hepatocellular carcinoma. Hepatogastroentero-logy 1999;46:320811.

18. Nguyen MH, Garcia RT, Simpson PW, Wright TL, KeeffeEB. Racial differences in effectiveness of alpha-fetopro-



tein for diagnosis of hepatocellular carcinoma in hepa-titis C virus cirrhosis. Hepatology 2002;36:4107.

19. Shiraki K, Takase K, Tameda Y, Hamada M, Kosaka Y,Nakano T. A clinical study of lectin-reactive alpha-feto-protein as an early indicator of hepatocellular carcinomain the follow-up of cirrhotic patients. Hepatology 1995;22:8027.

20. Hayashi K, Kumada T, Nakano S, Takeda I, Sugiyama K,Kiriyama S, et al. Usefulness of measurement of Lensculinaris agglutinin-reactive fraction of alpha-fetoproteinas a marker of prognosis and recurrence of smallhepatocellular carcinoma. Am J Gastroenterol 1999;94:302833.

21. Kumada T, Nakano S, Takeda I, Kiriyama S, Sone Y,Hayashi K, et al. Clinical utility of Lens culinaris agglu-tinin-reactive alpha-fetoprotein in small hepatocellularcarcinoma: special reference to imaging diagnosis. JHepatol 1999;30:12530.

22. Wang CS, Lin CL, Lee HC, Chen KY, Chiang MF, ChenHS, et al. Usefulness of serum des-gamma-carboxyprothrombin in detection of hepatocellular carcinoma.World J Gastroenterol 2005;11:61159.

23. Bruix J, Sherman M, Llovet JM, Beaugrand M, LencioniR, Burroughs AK, et al. Clinical management of hepato-cellular carcinoma. Conclusions of the Barcelona 2000EASL conference. J Hepatol 2001;35:42130.

24. Kuromatsu R, Tanaka M, Shimauchi Y, ShimadaM, Tani-kawa K, Watanabe K, et al. Usefulness of ED036 kit formeasuring serum PIVKA-II levels in small hepatocellularcarcinoma. J Gastroenterol 1997;32:50712.

25. Mita Y, Aoyagi Y, Yanagi M, Suda T, Suzuki Y, AsakuraH. The usefulness of determining des-gamma-carboxyprothrombin by sensitive enzyme immunoassay in theearly diagnosis of patients with hepatocellular carcino-ma. Cancer 1998;82:16438.

26. Okuda H, Nakanishi T, Takatsu K, Saito A, Hayashi N,Watanabe K, et al. Measurement of serum levels of des-gamma-carboxy prothrombin in patients with hepato-cellular carcinoma by a revised enzyme immunoassaykit with increased sensitivity. Cancer 1999;85:8128.

27. Izuno K, Fujiyama S, Yamasaki K, Sato M, Sato T. Earlydetection of hepatocellular carcinoma associated withcirrhosis by combined assay of des-gamma-carboxyprothrombin and alpha-fetoprotein: a prospective study.Hepatogastroenterology 1995;42:38793.

28. Suzuki M, Shiraha H, Fujikawa T, Takaoka N, Ueda N,Nakanishi Y, et al. Des-gamma-carboxy prothrombin isa potential autologous growth factor for hepatocellularcarcinoma. J Biol Chem 2005;280:640915.

29. Fujiyama S, Morishita T, Sagara K, Sato T, Motohara K,Matsuda I. Clinical evaluation of plasma abnormal pro-thrombin (PIVKA-II) in patients with hepatocellular car-cinoma. Hepatogastroenterology 1986;33:2015.

30. Saitoh S, Ikeda K, Koida I, Tsubota A, Arase Y, ChayamaK, et al. Serum des-gamma-carboxyprothrombin con-centration determined by the avidin-biotin complexmethod in small hepatocellular carcinomas. Cancer1994;74:291823.

31. Ishii M, Gama H, Chida N, Ueno Y, Shinzawa H, TakagiT, et al. Simultaneous measurements of serum alpha-fetoprotein and protein induced by vitamin K absencefor detecting hepatocellular carcinoma. South TohokuDistrict Study Group. Am J Gastroenterol 2000;95:103640.

32. Toyoda H, Kumada T, Kiriyama S, Sone Y, Tanikawa M,Hisanaga Y, et al. Prognostic significance of simultane-ous measurement of three tumor markers in patientswith hepatocellular carcinoma. Clin Gastroenterol Hepa-tol 2006;4:1117.

Documents

Development of Des-_-carboxy Prothrombin