Performance evaluation of serum PIVKA‐II measurement using HISCL‐5000 and a method comparison of HISCL‐5000, LUMIPULSE G1200, and ARCHITECT i2000

Abstract Background Protein induced by vitamin K antagonist‐II (PIVKA‐II), in addition to alpha‐fetoprotein, is a useful tumor marker for diagnosis of hepatocellular carcinoma (HCC). We evaluated the analytical performance of the HISCL‐5000 analyzer (Sysmex Corporation) in the measurement of serum PIVKA‐II. Methods We evaluated the precision and linearity of PIVKA‐II assays using the HISCL‐5000 analyzer. Methods using HISCL‐5000, LUMIPULSE G1200 (Fujirebio Diagnostics), and ARCHITECT i2000 (Abbott Diagnostics) were compared according to the guidelines of the Clinical and Laboratory Standards Institute. A total of 501 subjects (median age 59 years, age range 24‐90 years) were enrolled. Among them, 335 were HCC patients, 46 were patients with non‐HCC liver disease, and 120 were healthy individuals. Non‐HCC liver disease included liver cirrhosis, chronic hepatitis, HBV or HCV carrier, hepatic adenoma, and intrahepatic cholangiocarcinoma. Results Repeatability (%CV) in low‐ and high‐level controls and pooled serum was 2.81%‐10.30%, and within‐laboratory precision was 4.24%‐8.86%. In a linearity test, the coefficient of determination (R 2) was 0.9957, ranging from 11 to 69 897 mAU/mL. In comparison, the coefficient of correlation (r) was 0.9561‐0.9644, agreement was 93.4%‐97.6%, and the κ value was 0.855‐0.945 among the three analyzers. About 99.2% of healthy individuals and 84.8% of non‐HCC liver disease patients were below the cutoff value (40 mAU/mL) on HISCL‐5000. Conclusions A PIVKA‐II assay using HISCL‐5000 showed acceptable analytical performance including precision, linearity, and method comparison. This indicates that HISCL‐5000 can be potentially helpful in clinical laboratories.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is the fourth most common cancer in men and the sixth in women; in Korea, it is the second most common cause of death from cancer in men and the fourth in women. 1 HCC has a mortality rate higher than that of other cancers in patients in their 40s and 50s. It is important to detect early-stage HCC in high-risk groups such as those with HBV infection, HCV infection, or cirrhosis. Several serum markers for diagnosing liver fibrosis or cirrhosis are known, and the diagnosis of HCC is based on pathology or imaging studies. 2,3 Regular surveillance is warranted in these high-risk groups. 2,4,5 HCC surveillance programs for high-risk groups have been performed using ultrasound and serum alpha-fetoprotein (AFP) level in Korea. 1 However, serum AFP level is normal in some small HCC and can be nonspecifically elevated in patients with non-HCC liver disease. 2 A recent Japanese guideline indicates the combined use of AFP and protein induced by vitamin K antagonist-II (PIVKA-II) for diagnosis of HCC. 6 Several studies also have reported the clinical usefulness of PIVKA-II. [7][8][9] PIVKA-II, which is also referred to as des-gamma-carboxy prothrombin, is widely used as a tumor marker in addition to AFP for diagnosis of HCC. PIVKA-II is an abnormal prothrombin produced in the absence of vitamin K or when its activity is decreased in liver cells. Normally, 10 glutamic acids (Glu) in the N-terminal domain are converted to γ-carboxyglutamic acids (Gla) by a vitamin K-dependent carboxylase. In the presence of vitamin K deficiency, all or some of the 10 Glu cannot be converted to Gla, and abnormal prothrombin can be secreted into the blood. 10,11 In 1984, Liebman et al 12 first reported serum PIVKA-II level to be significantly elevated in HCC patients. Many studies have reported that PIVKA-II is a good and effective biomarker for detection and surveillance of HCC. The incidence of HCC is highest in East Asia, sub-Saharan Africa, and Melanesia, where around 83% of cases occur. 13 PIVKA-II tests have routinely been used to screen for HCC in addition to ultrasound in Japan, whereas they are not recommended in Europe and America. 14,15 PIVKA-II has been considered an essential marker for HCC surveillance in Asia. 16 The present study aimed to evaluate the analytical performance of serum PIVKA-II measurement using an HISCL-5000 analyzer (Sysmex Corporation) and to compare concentrations of PIVKA-II measured using HISCL-5000, LUMIPULSE G1200, and ARCHITECT i2000.

| Study population
We collected serum from 501 individuals between October 2017 and December 2017 at Samsung Medical Center. Among a total of 501 subjects, 335 had HCC, 46 had non-HCC liver disease, and 120 were healthy individuals who attended a regular health checkup (

| Instruments
We evaluated the basic performance of PIVKA-II assays using the HISCL-5000 analyzer, a fully automated immunochemistry analyzer that employs a chemiluminescence enzyme immunoassay (CLEIA) methodology with a two-step sandwich immunoassay. The primary antibody was anti-PIVKA-II mouse monoclonal antibody (MU-3 antibody), and the secondary antibody was anti-prothrombin mouse  were performed according to the manufacturers' instructions.

| Precision
Precision was assessed using two levels of quality control material and pooled serum, according to the Clinical and Laboratory Standards Institute's (CLSI) EP05-A3 guideline. 17 High-and low-level quality control substances for PIVKA-II were provided by the manufacturer. Repeatability and within-laboratory precision (%CV) were assessed by measuring twice each day for 20 days, and each test value was determined as the mean value of the two measurements.

| Linearity
Linearity was evaluated according to the CLSI EP06-A guideline. 18 The high-and low-concentration control materials that were close to the upper and lower limits of the measurement range, respectively, were used. Linearity was evaluated at mixture ratios of 4:0, 3:1, 2:2, 1:3, and 0:4. The test was repeated four times for each of the five concentrations, and linearity was evaluated as the coefficient of determination (R 2 ).

| Comparison
The comparison test was performed to compare the HISCL-5000 assay to the LUMIPULSE G1200 assay and ARCHITECT i2000 assay,

| D ISCUSS I ON
Enzyme-linked immunosorbent assay (ELISA) using an anti-PIVKA-II monoclonal antibody has been widely used in clinical laboratories. 16,20 Fully automated analyzers using various methodologies such as CLEIA or CMIA-for example, LUMIPULSE G1200 and ARCHITECT i2000-have been introduced and used clinically, and their analytical performances have been evaluated. 16,21 Recently, Sysmex Corporation has released a newly developed PIVKA-II assay kit using the HISCL-5000 analyzer.
This study showed that serum PIVKA-II by HISCL-5000 had acceptable precision and linearity and was comparable to LUMIPULSE For 36 patients, discordant results in any of the three analyzer pairs were mostly due to PIVKA-II values around or near the cutoff value (40 mAU/mL), and most of them (31/36) were from the HCC group.
Larger differences were observed at higher concentrations between LUMIPULSE G1200 and HISCL-5000 and between ARCHITECT i2000 and HISCL-5000. This could be caused by use of different antibodies or interference of endogenous substances in the different patient samples. Different antibodies recognize different amino acid residues in the Gla domain, 23 and conformational change of the antigens may be responsible for the differences.
As shown in Figure 4, there was no significant difference in median serum level of PIVKA-II between the HCC group and the non-HCC liver disease group (Mann-Whitney, P = 0.150). Serum PIVKA-II level may be elevated in not only HCC but also liver cirrhosis, because of alterations in vitamin K production secondary to cholestasis, malnutrition, or use of medication. 24,25 In this study, seven of the 46 non-HCC liver disease patients were above the cutoff value (40 mAU/mL), and they were all liver cirrhosis patients. Although they showed higher values, serum PIVKA-II level range at higher concentrations in the HCC group was much wider than that of the with a baseline PIVKA-II level >200 mAU/mL showed a significant correlation between overall survival and PIVKA-II response defined as a reduction greater than 50% from baseline after TACE.
Yamamoto et al 22 have reported that positive PIVKA-II values became negative at 6 months posthepatectomy in 99.6% of patients.
In conclusion, the PIVKA-II assay using HISCL-5000 showed acceptable analytical performance including precision, linearity, and method comparison, and there was a high degree of agreement among the three analyzers. This indicates that HISCL-5000 can be potentially helpful in clinical laboratories.

ACK N OWLED G M ENTS
The reagents used in this research were supplied from Sysmex Corporation and Abbott Diagnostics.

CO N FLI C T O F I NTE R E S T
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AUTH O R CO NTR I B UTI O N S
EK and HP contributed equally to this work. HP conceived and designed the study. MR, EK, and HP performed the experiments and analyzed the data. MR wrote the manuscript. EK and HP reviewed and modified the manuscript.

E TH I C A L A PPROVA L
This study was approved by the Ethics Committee of Samsung Medical Center (reference number: 2017-07-153-001). Guarantor: Hyung-Doo Park.