Article in press - uncorrected proof

Clin Chem Lab Med 2008;46(3):411416
2008 by Walter de Gruyter Berlin New York. DOI 10.1515/CCLM.2008.078

2007/424

Development of des-g-carboxy prothrombin (DCP) measuring

reagent using the LiBASys clinical analyzer

Isao Yamaguchi*, Kenji Nakamura, Hiromichi

Kitano, Yoshie Masuda, Futoshi Kanke, Shinzo
Kobatake and Shinji Satomura

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

ting (1). It is formed in the liver, when the 10 glutamic

acid (Glu) residues in the amino terminal in its precursor are converted to g-carboxyglutamic acid (Gla)
by a vitamin K-dependent carboxylase in the posttranslational process (2). When all or some of the 10
Glu residues are not converted to Gla, an immature
form of prothrombin is secreted into the blood,
termed des-g-carboxy prothrombin (DCP) or protein
induced by vitamin K absence or antagonist-II (PIVKAII) (3).
Liebman et al. showed that DCP is a serum tumor
marker for primary hepatocellular carcinoma (HCC)
(4). DCP has since been investigated and used as a
specific tumor marker for HCC in Japan. a-Fetoprotein
(AFP) is an oncofetal glycoprotein that has been used
as a tumor marker for HCC. However, AFP is not specific to HCC. Benign liver diseases, such as chronic
hepatitis and cirrhosis, are known to increase AFP
concentration in blood. Recently, the Lens culinaris
agglutinin-reactive a-fetoprotein ratio (AFP-L3), which
has an a16 fucose residue appended to N-acetylglucosamine at the reducing end, has been reported to
be a specific marker for HCC. In Japan, DCP and AFPL3%, the ratio of AFP-L3 to total AFP, have been widely and routinely used as serum markers for HCC. It
has been reported that the rate of detection of small
HCC is improved by a combination assay with DCP
and AFP-L3 and that these markers are complementary and useful for the diagnosis and evaluation of
small HCC when measured simultaneously (5, 6).
We developed the LiBASys clinical analyzer based
on the liquid binding assay (LBA) method (7, 8). In the
LBA method, the antigen-antibody reaction is formed
in the liquid phase to enable optimization of the antibody concentration and greatly shortens antigen-antibody reaction times. We have already established
simultaneous determination of both total AFP and
AFP-L3 with the LiBASys (8). As the combination of
DCP and AFP-L3% measurements improves HCC
detection, a DCP assay was developed on the LiBASys
instrument, making single-platform measurement of
DCP and AFP-L3% possible.

Introduction

Materials and methods

Prothrombin is a vitamin K-dependent coagulation

factor that is activated to thrombin during blood clot-

Materials

New Diagnostics Business and Technology

Development Department, Wako Pure Chemical
Industries Ltd., Hyogo, Japan

Abstract
Background: Des-g-carboxy prothrombin (DCP), Lens
culinaris agglutinin-reactive a-fetoprotein ratio (AFPL3) and total a-fetoprotein (AFP) are tumor markers
useful for diagnosing and determining the prognosis
of hepatocellular carcinoma (HCC). There is a real
need for measurement of these three markers on a
one-assay platform.
Methods: A method of DCP measurement in human
serum was developed using liquid binding assay
(LBA), which enables rapid antigen-antibody reaction
and bound/free separation on the LiBASys clinical
analyzer.
Results: The dilution curve for DCP was linear up to
500 ng/mL. The limit of detection of DCP concentration was 0.5 ng/mL. Intra- and inter-assay coefficients of variation of DCP were 0.7%2.4% and
2.2%6.5%, respectively. This method was free from
interference by hemoglobin, bilirubin, ditaurobilirubin, intrafat, ascorbate, galactose, glucose and rheumatoid factor. The analytical recoveries of DCP added
to serum were 91.7%108.2%. DCP concentration
measured with the LBA method was linear and was
significantly correlated with that measured with the
ELISA method.
Conclusions: The LiBASys clinical analyzer made
possible measurement of the complementary tumor
markers, HCC, total AFP, AFP-L3 and DCP.
Clin Chem Lab Med 2008;46:4116.

*Corresponding author: Isao Yamaguchi, New Diagnostics

Business and Technology Development Department, Wako
Pure Chemical Industries, Ltd., 6-1 Takada-cho, Amagasaki,
Hyogo, 661-0963, Japan
Phone: q81-6-6499-9109, Fax: q81-6-6499-1524,
E-mail: yamaguchi.isao@wako-chem.co.jp
Received September 10, 2007; accepted November 23, 2007;
previously published online February 6, 2008

All serum samples were stored below y808C until measurement. Sulfated tyrosine-pentamer (YS5) was synthesized and
was labeled with the single binding moiety (Fab9) of antiprothrombin monoclonal antibody (mouse) to prepare Fab9YS5 antibody. Peroxidase (POD) was from Toyobo (Osaka,
Japan) and was labeled with the Fab9 of anti-DCP monoclonal antibody (mouse) to prepare Fab9-POD. Anti-DCP monoclonal antibody is a highly specific antibody to DCP prepared

Article in press - uncorrected proof

412

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

using a synthetic peptide (1135 positions) as the antigen

and the cell line SP2/O as a myeloma. Serum containing
human anti-mouse antibody (HAMA) was from Roche (Basel,
Switzerland). DCP concentration was measured using an
enzyme-linked immunosorbent assay method (Eitest PIVKAII, Eisai, Tokyo, Japan). AFP-L3 and total AFP were measured
using the LBA method (LBA AFP-L3, Wako, Osaka, Japan).
Purified DCP was prepared using a thermal decarboxylation
procedure and by using affinity chromatography with antiDCP monoclonal antibody.

Determination of DCP concentration

The purified DCP was isolated from decarboxylated prothrombin by heating and was purified by immuno-affinity
chromatography with a monoclonal antibody specific for
DCP (clone PI-1). Clone PI-1 does not recognize normal prothrombin. The affinity column was prepared using NHS-activated Sepharose 4 Fast Flow Lab Packs (GE Healthcare, Little
Chalfont, UK). DCP contents of the column eluates in the
purification steps were monitored by determining absorbance at 280 nm. The purified DCP was tested for a single
protein band on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). The protein concentration of
the purified DCP (ng/mL) was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the
manufacturers protocols. The DCP concentration of purified
DCP wmilli-arbitrary unit (mAU)/mLx was determined using
an Eitest PIVKA-II. We converted from the conventional unit
(AU) to the SI unit (g) and calculated the DCP conversion
factor from measurement data.

Apparatus
Automated assay of DCP concentration was performed with
the 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) separation
and an optical assembly for fluorophotometric detection.
With the LiBASys, the antigen-antibody reaction is performed in the liquid phase, and B/F separation is rapidly
performed using column chromatography without a solid
phase. The outer cuvettes are incubated at 378C. The inner
cuvettes and sample carousel are cooled below 108C.

5 min. Fab9-POD and Fab9-YS5 bind to DCP and form immune

complexes. After incubation, 80 mL of the reaction mixture
is introduced into an anion-exchange column. The immune
complexes have affinity for the column, but none for free
Fab9-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 the
column with Elution buffer C. A total of 900 mL of eluate is
poured into outer cuvettes incubated at 378C. In addition,
100 mL of substrate solution is added to eluate containing
immune complexes, and the POD activity of the immune
complexes is then measured. POD activity is determined as
the increase in fluorescence intensity. These values are compared to fluorescence intensities of known standards for DCP
concentration to obtain the DCP concentrations of samples.

Linearity
The dilution curve was prepared by plotting the measured
DCP concentration on the y-axis vs. the dilution ratio of DCP
on the x-axis. A high DCP value sample was prepared by
spiking DCP to a pool serum and diluted. Saline was used
for sample dilution.

Limit of detection
The minimum limit of detection was determined in eight replicates of samples containing known amounts of DCP. The
DCP concentration that corresponded to the zero standard
plus two times the standard deviation (SD) was determined
to be the limit of detection.

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

Recovery

Reagents
The reagent consisted of Reagent, Substrate 1 and Substrate
2. The Reagent consists of 39.7 nmol/L Fab9-POD and
235 nmol/L Fab9-YS5 in 50 mmol/L N-(2-acetamido)-2aminoethanesulfonate buffer, pH 6.5. Opened Reagent can
be used for 10 days at 2108C on the LiBASys. Substrate 1
consists of 320 mmol/L 4-acetoamidophenol in 2-propanol.
Substrate 2 consists of 40 mmol/L hydrogen peroxide in
15 mmol/L citrate buffer, pH 5.5. One bottle of Substrate 1 is
added to one bottle of Substrate 2, mixed well, and used as
the working substrate solution. Mixed substrate solution is
stable for 10 days when stored at 2108C. Wash solution consists of 1.0 mol/L citrate. Elution buffers A, B and C consist
of 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 prepared with human prothrombin decarboxylated by heating.
(9).

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

The recovery of DCP from serum samples was measured by

adding known amounts of DCP to serum samples.

Interference studies
Several endogenous substances, including hemoglobin, bilirubin, ditaurobilirubin, intrafat, ascorbate, galactose, glucose, rheumatoid factor and HAMA were added to serum
samples to assess interference. Serum samples containing
DCP without interference substances were measured as a
control. Recovery of DCP using HAMA was examined to
assess interference by adding known amounts of DCP to
HAMA or normal serum.

Comparison of methods
Serum samples were measured with the LBA method and
the ELISA method. The DCP concentrations measured with
the LBA method were compared with those measured with
the ELISA method.

Article in press - uncorrected proof

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

Results

mediate concentration and Figure 2C for low

concentration.

Determination of DCP concentration

Four lots of purified DCP samples were prepared
using a thermal decarboxylation procedure and purified by using immune affinity chromatography with
anti-DCP monoclonal antibody. Each sample was confirmed using SDS-PAGE. SDS-PAGE was performed
by the method of Laemmli (10) with 7.5% running gel.
Gel was stained with Coomassie Brilliant Blue R-250
for proteins. On SDS-PAGE, every sample migrated as
a single band, indicating pure DCP (Figure 1).
DCP and protein concentrations of sample were
determined using the BCA protein assay kit (ng/mL)
and the DCP detection kit (mAU/mL). The DCP conversion factor to convert from the conventional unit
(AU) to the SI unit (g) for each sample was calculated
from measurement data. The DCP conversion factors
for mAU to ng were 0.016 to 0.023 ng/mAU. The mean
value of samples was 0.019 ng/mAU, with a SD of
0.003. For the reasons indicated above, the DCP
conversion factor of mAU to ng was fixed at 0.019
ng/mAU (Table 1).
Linearity
Typical dilution curves for the presented DCP assay
are shown in Figure 2AC. Figure 2A is the dilution
curve for high concentration, Figure 2B for inter-

Limit of detection
The dilution curve was linear between 0 and 500
ng/mL. The analytical limit of detection of DCP concentration was estimated as the dose equivalent to
the mean of eight replicates of the zero standard plus
2 SD. The DCP concentration that corresponded to
this signal was calculated to be 0.50 ng/mL (Figure 3).
Precision, recovery and interference studies
The intra-assay (21 replicates) and inter-assay (21
replicates for 28 days) CVs of DCP concentration were
0.7%2.4% and 2.2%6.5%, respectively (Table 2).
The analytical recovery of DCP added to serum
ranged from 91.7% to 108.2% (Table 3). Endogenous
substances, including hemoglobin (02 g/L), bilirubin
(04 mg/L), ditaurobilirubin (02 mg/L), intrafat
(0%2%), ascorbate (05 mg/L), galactose (020
mg/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 ranged
from 87.1% to 109.1%. HAMA did not interfere with
assay results.
Comparison of methods
DCP concentrations measured by the LBA method
were linear and were significantly correlated with
those measured by the ELISA method. The regression
formula for DCP was ys0.0194xy0.0468 (Figure 4;
ysDCP concentration by LBA method, ng/mL; xsDCP
concentration by ELISA method, mAU/mL; ns49;
rs0.9956).

Discussion

Figure 1 SDS-PAGE of purified DCP samples.

SDS-polyacrylamide slab gel electrophoresis was performed
by the method of Laemmli with 7.5% running gel. Gels were
stained with Coomassie Brilliant Blue R-250 for proteins.

HCC is one of the most common cancers in the world.

Most HCCs develop in cirrhotic liver, and patients with
cirrhosis comprise a high-risk group for the development of HCC (11). Total AFP, AFP-L3 and DCP have
been widely used clinically as tumor markers of HCC
in Japan (4, 1214). Total AFP is a commonly used
tumor marker of HCC, exhibits acceptable sensitivity
and generally reflects the amount of tumor mass (6).
However, AFP sometimes increases not only in HCC
patients but also in cirrhosis patients (15, 16), and
does not increase in 35%45% of HCC patients (17,
18). It is thus difficult to distinguish early-stage HCC

Table 1 Results of determination of DCP conversion factor.

DCP lot

DCP, mAU/mL
Protein, ng/mL
DCP conversion factor, ng/mAU

4,723,000
92,000
0.019

4,950,000
111,600
0.023

3,814,000
64,100
0.017

4,000,000
64,500
0.016

SD

Mean

0.003

0.019

Article in press - uncorrected proof

414

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

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 dilution
curve was linear between 0 and 500 ng/mL. (A) High concentration of DCP, (B) intermediate concentration of DCP and (C) low
concentration of DCP.

Figure 3 Limit of detection.

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

patients from cirrhosis patients using total AFP alone.

AFP-L3 is the ratio of the structure of carbohydrate
chain changes according to the cancerization of cells
and is the percentage of AFP with affinity for Lens
Table 2 Precision of DCP.
Sample

1
2
3
4
5
6

Mean
DCP, ng/mL

CV (%)
Intra-assay

Inter-assay

1.0
2.4
8.2
57.4
190.6
453.2

1.9
1.6
2.1
0.7
1.8
2.4

6.5
3.3
2.2
2.5
2.4
2.5

Values are given as means across all LiBASys analyzers.

culinaris agglutinin against total AFP. It has been

reported that AFP-L3 is highly specific for HCC and
yields positive results earlier than diagnosis of HCC
by imaging modalities (12, 15, 19). In addition, it has
been reported that AFP-L3 is useful in determining the
prognosis after treatment of HCC, as well as the biological malignancy of HCC (20, 21). DCP is an immature form of prothrombin, where all or some of the 10
Glu residues are not converted to Gla. DCP showed
strong correlation with tumor size of HCC and can be
used to detect early-stage HCC (2226). Izuno et al.
reported a correlation between DCP level and the
existence of multiple HCCs (27). Suzuki et al. suggested that DCP concentration was a useful predictor
of portal venous invasion in patients with HCC. It has
also been reported that it is possible that DCP acts as
an autologous mitogen and stimulates the proliferation of HCC (28).
Because no correlations have been found between
DCP and total AFP (2931) or between DCP and AFPL3 (5, 6) in HCC patients, the combination assay with
AFP-L3 and DCP was used to measure complementary markers. It has been reported that a combination
assay improves the sensitivity in detection of HCC (5,
6, 21, 30) and is useful for follow-up of cirrhosis
patients (6). Toyoda et al. suggested that simultaneous measurement of these tumor markers could be
useful for the evaluation of tumor progression, prediction of patient outcome and determination of treatment efficacy (32).
However, no clinical analyzer measuring both AFPL3 and DCP has been available. We have already
developed a simultaneous determination reagent for
total AFP and AFP-L3 with the LiBASys clinical analyzer (8). In addition, a reagent for determination of
DCP concentration in serum on the LiBASys has been
established with excellent performance. Using the
LiBASys, the combination assay of AFP-L3 and DCP
was easily performed. The LiBASys could be useful
for the early detection and determination of the prognosis of HCC.

Article in press - uncorrected proof

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

Table 3 Recovery of DCP.

Sample

DCP,
ng/mL

2.1

14.9

108.2

DCP added,
ng/mL

DCP measured,
ng/mL

Obtained,
ng/mL

Recovery, %

1.2
2.3
5.2
8.7
1.4
4.7
9.6
18.4
36.7
83.1
193.2
346.3

3.2
4.4
7.3
11.2
16.3
19.7
24.7
34.7
54.6
171.2
387.3
713.9

1.1
2.3
5.2
9.1
1.4
4.8
9.9
19.8
39.7
88.1
194.1
367.6

91.7
100.0
100.0
104.6
100.0
102.1
103.1
107.6
108.2
106.0
100.5
106.2

6.

7.

8.

9.

10.
Figure 4 Comparison of DCP concentrations determined by
the LBA method and the ELISA method.
DCP concentrations measured by the LBA method were significantly correlated with those measured by the ELISA
method. The ELISA method (Eitest PIVKA-II, Eisai, Tokyo,
Japan) allows measurement of DCP in the range of
102000 mAU/mL.

References
1. Barton PG, Jackson CM, Hanahan DJ. Relationship
between factor V and activated factor X in the generation
of 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 metabolism 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) prothrombin as a serum marker of primary hepatocellular carcinoma. N Engl J Med 1984;310:142731.
5. Sassa T, Kumada T, Nakano S, Uematsu T. Clinical utility
of simultaneous measurement of serum high-sensitivity
des-gamma-carboxy prothrombin and Lens culinaris
agglutinin A-reactive alpha-fetoprotein in patients with

11.
12.

13.

14.

15.

16.

17.

18.

small hepatocellular carcinoma. Eur J Gastroenterol

Hepatol 1999;11:138792.
Shimauchi Y, Tanaka M, Kuromatsu R, Ogata R, Tateishi
Y, Itano S, et al. A simultaneous monitoring of Lens
culinaris agglutinin A-reactive alpha-fetoprotein and
des-gamma-carboxy prothrombin as an early diagnosis
of hepatocellular carcinoma in the follow-up of cirrhotic
patients. Oncol Rep 2000;7:24956.
Nakamura K, Satomura S, Matsuura S. Liquid-phase
binding assay of human chorionic gonadotropin using
high-performance liquid chromatography. Anal Chem
1993;65:6136.
Yamagata Y, Shimizu K, Nakamura K, Henmi F, Satomura S, Matsuura S, et al. Simultaneous determination
of percentage of Lens culinaris agglutinin-reactive alphafetoprotein and alpha-fetoprotein concentration using
the LiBASys clinical auto-analyzer. Clin Chim Acta 2003;
327:5967.
Bajaj SP, Price PA, Russell WA. Decarboxylation of
gamma-carboxyglutamic acid residues in human prothrombin. J Biol Chem 1982;257:372631.
Laemmli UK. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature
1970;227:6805.
Okuda K. Early recognition of hepatocellular carcinoma.
Hepatology 1986;6:72938.
Abelev GI. Production of embryonal serum alpha-globulin by hepatomas: review of experimental and clinical
data. Cancer Res 1968;28:134450.
Taketa K, Endo Y, Sekiya C, Tanikawa K, Koji T, Taga H,
et al. A collaborative study for the evaluation of lectinreactive alpha-fetoproteins in early detection of hepatocellular carcinoma. Cancer Res 1993;53:541923.
Yamashita F, Tanaka M, Satomura S, Tanikawa K. Prognostic significance of Lens culinaris agglutinin A-reactive
alpha-fetoprotein in small hepatocellular carcinomas.
Gastroenterology 1996;111:9961001.
Alpert E, Feller ER. Alpha-fetoprotein (AFP) in benign liver disease. Evidence that normal liver regeneration does
not induce AFP synthesis. Gastroenterology 1978;74:
8568.
Sato Y, Nakata K, Kato Y, Shima M, Ishii N, Koji T, et al.
Early recognition of hepatocellular carcinoma based on
altered profiles of alpha-fetoprotein. N Engl J Med 1993;
328:18026.
Peng YC, Chan CS, Chen GH. The effectiveness of serum
alpha-fetoprotein level in anti-HCV positive patients for
screening hepatocellular carcinoma. Hepatogastroenterology 1999;46:320811.
Nguyen MH, Garcia RT, Simpson PW, Wright TL, Keeffe
EB. Racial differences in effectiveness of alpha-fetopro-

Article in press - uncorrected proof

416

19.

20.

21.

22.

23.

24.

25.

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

tein for diagnosis of hepatocellular carcinoma in hepatitis C virus cirrhosis. Hepatology 2002;36:4107.
Shiraki K, Takase K, Tameda Y, Hamada M, Kosaka Y,
Nakano T. A clinical study of lectin-reactive alpha-fetoprotein as an early indicator of hepatocellular carcinoma
in the follow-up of cirrhotic patients. Hepatology 1995;
22:8027.
Hayashi K, Kumada T, Nakano S, Takeda I, Sugiyama K,
Kiriyama S, et al. Usefulness of measurement of Lens
culinaris agglutinin-reactive fraction of alpha-fetoprotein
as a marker of prognosis and recurrence of small
hepatocellular carcinoma. Am J Gastroenterol 1999;94:
302833.
Kumada T, Nakano S, Takeda I, Kiriyama S, Sone Y,
Hayashi K, et al. Clinical utility of Lens culinaris agglutinin-reactive alpha-fetoprotein in small hepatocellular
carcinoma: special reference to imaging diagnosis. J
Hepatol 1999;30:12530.
Wang CS, Lin CL, Lee HC, Chen KY, Chiang MF, Chen
HS, et al. Usefulness of serum des-gamma-carboxy
prothrombin in detection of hepatocellular carcinoma.
World J Gastroenterol 2005;11:61159.
Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni
R, Burroughs AK, et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona 2000
EASL conference. J Hepatol 2001;35:42130.
Kuromatsu R, Tanaka M, Shimauchi Y, Shimada M, Tanikawa K, Watanabe K, et al. Usefulness of ED036 kit for
measuring serum PIVKA-II levels in small hepatocellular
carcinoma. J Gastroenterol 1997;32:50712.
Mita Y, Aoyagi Y, Yanagi M, Suda T, Suzuki Y, Asakura
H. The usefulness of determining des-gamma-carboxy
prothrombin by sensitive enzyme immunoassay in the
early diagnosis of patients with hepatocellular carcinoma. Cancer 1998;82:16438.

26. Okuda H, Nakanishi T, Takatsu K, Saito A, Hayashi N,

Watanabe K, et al. Measurement of serum levels of desgamma-carboxy prothrombin in patients with hepatocellular carcinoma by a revised enzyme immunoassay
kit with increased sensitivity. Cancer 1999;85:8128.
27. Izuno K, Fujiyama S, Yamasaki K, Sato M, Sato T. Early
detection of hepatocellular carcinoma associated with
cirrhosis by combined assay of des-gamma-carboxy
prothrombin 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 is
a potential autologous growth factor for hepatocellular
carcinoma. J Biol Chem 2005;280:640915.
29. Fujiyama S, Morishita T, Sagara K, Sato T, Motohara K,
Matsuda I. Clinical evaluation of plasma abnormal prothrombin (PIVKA-II) in patients with hepatocellular carcinoma. Hepatogastroenterology 1986;33:2015.
30. Saitoh S, Ikeda K, Koida I, Tsubota A, Arase Y, Chayama
K, et al. Serum des-gamma-carboxyprothrombin concentration determined by the avidin-biotin complex
method in small hepatocellular carcinomas. Cancer
1994;74:291823.
31. Ishii M, Gama H, Chida N, Ueno Y, Shinzawa H, Takagi
T, et al. Simultaneous measurements of serum alphafetoprotein and protein induced by vitamin K absence
for detecting hepatocellular carcinoma. South Tohoku
District 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 simultaneous measurement of three tumor markers in patients
with hepatocellular carcinoma. Clin Gastroenterol Hepatol 2006;4:1117.