Prospective study on the risk of hepatocellular carcinoma among hepatitis C virus-positive blood donors focusing on demographic factors, alanine aminotransferase level at donation and interaction with hepatitis B virus

Authors


Abstract

The risk for hepatocellular carcinoma (HCC) among asymptomatic hepatitis C virus (HCV) carriers is not well understood. A community-based prospective study was conducted for over 8 years by record linkage to the Osaka Cancer Registry. The subjects were 1,927 individuals who were positive for anti-HCV through screening for second-generation HCV antibody (passive hemagglutination assay: ≥ 212) in voluntary blood donation. The risk factors for HCC and interaction between HCV and hepatitis B virus (HBV) infection were evaluated by including additional blood donors: 2,519 individuals positive for hepatitis B virus surface antigen (HBsAg) alone, 25 positive for both anti-HCV and HBsAg, 150,379 negative for both anti-HCV and HBsAg. The incidence of HCC (/105 person-years) among the HCV-positive individuals increased with age in both genders, ranging from 68 to 1,306 among those aged 45–74 years. In the HCV-positive individuals, the cumulative risk of developing HCC between the ages of 40 and 74 year was 21.6% among males and 8.7% among females. A stepwise increase in risk was noted as the serum alanine aminotransferase level increased or serum cholesterol level at baseline decreased in multivariate Cox proportional hazard analysis. The 9-year cumulative incidence of HCC among individuals positive for HCV alone, those positive for HBsAg alone and those positive for both was 3.0%, 2.0% and 12.0%, respectively. The age-and-sex-adjusted rate ratio was 126, 102 and 572, respectively, when those negative for both were used as a reference. The results demonstrate an increased risk for HCC among asymptomatic HCV-positive individuals in Japan. Coinfection with HBV and HCV carried a superadditive risk for HCC. © 2004 Wiley-Liss, Inc.

Approximately 170 million persons worldwide have persistent infection with hepatitis C virus (HCV),1 which is a risk factor for hepatocellular carcinoma (HCC). Some patients with persistent HCV infection advance through incrementally severe stages of chronic hepatitis to cirrhosis, occasionally culminating in HCC, although the majority of persons with persistent HCV infection remain asymptomatic. The incidence of HCV-associated HCC is increasing in several developed countries, including the United States,2, 3 Italy,4 France4 and Japan.5 However, it is not well understood what proportion of asymptomatic HCV-positive persons will develop HCC during their lifetime.

Many prospective studies have been conducted that assessed the risk of severe liver disease, including HCC, among individuals with chronic HCV infection.6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Some studies were conducted in tertiary-care treatment facilities,6, 7, 8, 9, 10, 11 and the proportion of severe cases may have been overestimated due to referral bias. On the other hand, several follow-up studies reported that patients with chronic HCV infection have a very low risk of developing severe liver disease.12, 13, 14, 15, 16, 17 This result may have been obtained due to an insufficient length of follow-up to describe the full extent of adverse sequelae of HCV infection or because the subjects were too young to develop severe liver disease. In a cohort study of patients with non-A, non-B, transfusion-associated hepatitis over a period of almost 25 years, liver-related mortality was only 4.1%.18 As this cohort had high all-cause mortality probably resulting from the condition that required the original transfusion, the liver-related mortality reported in that study may have been underestimated. Studies performed in community-based settings have an advantage in clarifying the risk of HCC among asymptomatic HCV-positive persons, who comprise the majority of HCV-infected persons. However, several previous community-based prospective studies had a relatively small sample size.19, 20, 21 In another community-based prospective study, the subjects were persons who had been infected with HCV through injection drug use and who had high competing mortality.22

Screening blood donors for the antibody to HCV (anti-HCV) in the early 1990s revealed that there was a large group of apparently healthy individuals who were positive for anti-HCV. We conducted a prospective study to determine the absolute risk of developing HCC among persons who were incidentally detected of having HCV at the time of blood donation and to elucidate factors associated with the development of HCC among HCV-positive blood donors. This study was conducted on a large number of voluntary blood donors in Osaka, where there is a high incidence of liver cancer.23 The effect of the interaction between hepatitis B virus (HBV) and HCV infections on the risk for HCC was also evaluated.

MATERIAL AND METHODS

The subjects were selected from voluntary blood donors who gave 1,235,926 allogeneic blood donations at the Osaka Red Cross Blood Center between 1991 and 1993. Candidates for potential blood donors in Japan were described elsewhere.24 In brief, they must be between the ages of 16 and 64 years and have a hemoglobin level of 12 g/dl or higher. Potential blood donors are screened by a self-administered questionnaire regarding their present illnesses, history of liver disease, history of blood transfusion, sexual behaviors and so on. Those who meet the criteria in the preliminary screening may donate blood without compensation. Using the data files containing information on donors and the results of the serologic screening test, we identified 667,461 individual donors by the donor's birth date, sex, first name, family name and ABO blood type. From these individuals, we selected Osaka residents aged 40 years or older at the time of blood donation who were negative for antibodies to human T-cell lymphotropic virus type 1. Those who were 39 years or younger were excluded because the incidence of HCV-related HCC in the Japanese population younger than 40 years of age is very low and they would be unlikely to develop HCC during the 8- to 9-year follow-up we undertook. The serum alanine aminotransferase (ALT) level expressed in Karumen unit (KU), serum total cholesterol level (mg/dl) and ABO blood type were obtained from the data files to analyze whether they are risk factors for HCC. An individual was defined as having HCV infection if the titer of anti-HCV in a second-generation passive hemagglutination assay (PHA; Dainabot, Tokyo, Japan) was 212 or higher, because the positive predictive value for being HCV RNA-positive in using this cutoff point in Japanese blood donors was guaranteed,25 and it has been successfully used as the criterion for informing the blood donor his or her HCV carrier state in the Japanese blood center. This study included 1,927 anti-HCV-positive (212 ≤), HBsAg-negative individuals. Two thousand three hundred sixty-two individuals who had an anti-HCV titer between 25 and 211 were excluded from the study. An individual was considered to have HBV infection if positive for HB surface antigen (HBsAg) in a reverse passive hemagglutination assay (Japan Red Cross, Tokyo, Japan). To study the effect of the interaction between HCV and HBV infections on the development of HCC, this study also included 2,519 anti-HCV-negative (25 >), HBsAg-positive individuals; 25 individuals who were positive for both anti-HCV and HBsAg; and 150,379 individuals who were negative for both anti-HCV and HBsAg.

Follow-up

The subjects were followed using the records at the Osaka Cancer Registry, which covers all of the Osaka prefecture, with a population of 8.8 million in 1995. It registers cancer cases using reports from hospitals and clinics and death certificates collected from the Osaka prefectural government.23 Parameters used in the record linkage were sex, date of birth, address (city, ward, town and village) and first Chinese character of the family name. Resemblance was confirmed by one of the authors who was blinded to the screening data. The follow-up period was from the date of blood donation through the date of diagnosis of hepatocellular carcinoma or 31 December 2000. Subjects who remained unaffected by HCC were censored at the last date of follow-up. This protocol was approved by the ethics committees of both the Osaka Medical Center for Cancer and Cardiovascular Diseases and the Osaka Red Cross Blood Center.

Statistical analyses

The number of sex- and 5-year age-specific person-years of observation in the 1,927 anti-HCV-positive, HBsAg-negative blood donors was determined to obtain the demographically stratified incidence rate of HCC (absolute risk). Using the sex- and age-specific incidence rate, we calculated the cumulative risk26 for the development of HCC among the anti-HCV-positive, HBsAg-negative individuals between the ages of 40 and 74 years by sex. The Kaplan-Meier method was used to calculate the cumulative incidence of HCC, and the log-rank test was used to assess the statistical significance. Independent factors associated with the development of HCC were studied in a Cox proportional hazard model. To assess whether there is an interaction between HCV and HBV infections in the development of HCC and, if so, the nature of the interaction, the rate ratios (RRs) were estimated among subjects who were seropositive for both anti-HCV and HBsAg, among subjects seropositive for anti-HCV alone, as well as among subjects seropositive for HBsAg alone, using subjects seronegative for both anti-HCV and HBsAg as a reference. Statistical assessment of coinfection on the development of HCC was performed on the basis of an additive scale by estimating the synergy index.27 The 95% confidence interval of the synergy index was calculated using Rothman's modified regression model.28 If the value of the synergy index was significantly higher than 1, the effect of the joint exposures was considered to be superadditive. Data analyses were performed with the SAS/PC statistical package (SAS Institute, Cary, NC). All presented p-values are 2-sided; p < 0.05 was considered to indicate significance.

RESULTS

Characteristics and annual incidence rate of HCC among 1,927 anti-HCV-positive, HBsAg-negative blood donors

There were 987 males and 940 females among the anti-HCV-positive, HBsAg-negative subjects (Table I). Sixty-one percent of the males and 77% of the females had a normal ALT level of ≤ 29 KU. Fifty-six percent of the males and 31% of the females had a serum cholesterol level of ≤ 169 mg/dl. There was no significant difference in the distribution of ABO blood types between the males and females.

Table I. Baseline Characteristics of the 1,927 Anti-HCV-Positive, HBsAg-Negative Blood Donors
Characteristic at entryMale (n = 987)Female (n = 940)Total (n = 1,927)
  1. ALT: alanine amino transferase, KU: Karumen unit.

Age at blood donation (years)(%)(%)(%)
 40–44227 (23.0)171 (18.2)398 (20.7)
 45–49225 (22.8)202 (21.5)427 (22.2)
 50–54205 (20.8)241 (25.6)446 (23.1)
 55–59205 (20.8)213 (22.7)418 (21.7)
 60–64125 (12.7)113 (12.0)238 (12.4)
ALT (KU)   
 29 or lower597 (60.5)721 (76.7)1318 (68.4)
 30–59257 (26.0)168 (17.9)425 (22.1)
 60 or higher133 (13.5)51 (5.4)184 (9.5)
Cholesterol (mg/dl)   
 139 or lower192 (19.5)58 (6.2)250 (13.0)
 140–169363 (36.8)237 (25.2)600 (31.1)
 170–199282 (28.6)320 (34.0)602 (31.2)
 200 or higher150 (15.2)325 (34.6)475 (24.6)
ABO blood type   
 A377 (38.2)376 (40.0)753 (39.1)
 B209 (21.2)194 (20.6)403 (20.9)
 O304 (30.8)263 (28.0)567 (29.4)
 AB96 (9.7)107 (11.4)203 (10.5)

During the follow-up (mean, 99 months; 15,891 person-years), 53 subjects (44 males, 9 females) developed HCC. The annual incidence rate increased with advancing age among all subjects, ranging from 0 in the 40–44 year age group to 1,306 in the 70–74 year age group. Among the anti-HCV-positive, HBsAg-negative individuals, the cumulative risk of developing HCC between the ages of 40 and 74 years was 21.6% among males and 8.7% among females (Table II).

Table II. Incidence Rate of HCC Among the 1,927 Anti-HCV-Positive, HBsAg-Negative Blood Donors
Age (years)Male (n = 987)Female (n = 940)Total (n = 1,927)
Person-yearsHCCAnnual incidence rate (10−5)Person-yearsHCCAnnual incidence rate (10−5)Person-yearsHCCAnnual incidence rate (10−5)
  1. Using the sex- and-age-specific incidence rate, the cumulative risk26 of HCC between the ages of 40 and 74 years was calculated. The cumulative risk was 21.6% among males and 8.7% among females. Mean follow-up period (month) 98.4 ± 14.8 for male, 99.5 ± 10.0 for female, 99.0 ± 12.7 for total.

40–44583003460092900
45–491,65721211,274002,931268
50–541,81663301,8011563,6187194
55–591,736126911,98531513,72215403
60–641,513161,0571,59021263,10318580
65–69720797271722791,4379627
70–747111,4188311,21115321,306
 Total8,096445437,795911515,89153334

Factors associated with development of HCC among 1,927 anti-HCV-positive, HBsAg-negative blood donors

Compared with the subjects with normal ALT (≤ 29 KU), the subjects with elevated ALT (30–59 KU, 60 KU ≤) had significantly higher cumulative risk for developing HCC both among the males and among the females (log-rank test, p < 0.001 and p < 0.001, respectively; Fig. 1). All 9 female HCC cases developed beyond 52 months after the blood donation. The 9-year cumulative incidence of HCC among those in the 3 subgroups of ALT level (≤ 29 KU, 30–59 KU, 60 KU ≤) was 1.4%, 8.2% and 14.3%, respectively, among males, and 0.6%, 1.8% and 4.0%, respectively, among females (Fig. 1).

Figure 1.

Cumulative incidence of hepatocellular carcinoma among the male (a) and female (b) anti-HCV-positive, HBsAg-negative blood donors according to the serum ALT level (Karumen unit) at enrollment. The numbers at the bottom of each graph represent the number of patients with chronic HCV infection, categorized according to the serum ALT level, who were at risk for developing HCC at the indicated observation point.

Compared with the subjects of 40–49 years at the blood donation, the subjects of 50 years or older had a significantly higher risk of developing HCC (Table III). Females were significantly less likely to develop HCC than males (p = 0.011). Having an elevated ALT (ALT ≥ 30 KU) at the blood donation was a strong risk factor for developing HCC. A low serum cholesterol level at blood donation (≤ 139 mg/dl) was associated with high risk for developing HCC. In linear trend models, a stepwise increase in the risk for HCC was noted with an increase in the ALT level category (p < 0.0001) and decrease in the cholesterol level category (p < 0.001). Subjects with A, B, or O blood type tended to be more likely to develop HCC than those with AB blood type, although the differences were not significant (Table III).

Table III. Factors Associated with the Development of HCC in Blood Donors with Chronic Hepatitis C Infection According to Cox Proportional Hazard Analysis
VariablenHCCRate ratio95% CI
  • Cox proportional hazard analysis was performed on the 1,927 anti-HCV-positive, HBsAg-negative blood donors. Age (4 categories), sex, serum ALT level at blood donation (3 categories), serum cholesterol level at blood donation (4 categories) and ABO blood type were included as independent variables.

  • 1

    Significant stepwise increase in the risk of HCC was founded with an increase in the ALT level (p < 0.0001) and decrease in the cholesterol level (p < 0.001).

Age at blood donation, years    
 40–4982571.00 
 50–54446164.862.00–11.83
 55–59418184.471.85–10.80
 60–64238126.952.72–17.74
Sex    
 Male987441.00 
 Female94090.380.18–0.80
ALT level at blood donation1    
 29 KU or lower1318101.00 
 30–59425236.232.86–13.53
 60 KU or higher184209.544.23–21.49
Cholesterol level at blood donation1    
 200 mg/dl or higher47521.00 
 170–19960271.970.41–9.56
 140–169600223.750.86–16.31
 139 or lower250226.161.39–27.35
ABO blood type    
 AB20321.00 
 A753152.290.52–10.05
 B403112.780.62–12.59
 O567243.540.84–15.04

Interaction between HCV and HBV infections

Among the 2,519 anti-HCV-negative, HBsAg-positive subjects (1,532 males, 987 females; mean age at blood donation, 47.5 years), 45 developed HCC at a mean observation period of 104 months. Among the 25 subjects who were positive for both anti-HCV and HBsAg (16 males, 9 females; mean age at blood donation, 50.0 years), 3 developed HCC at a mean observation period of 86 months. The incidence of HCC was significantly higher among the subjects who were positive for both anti-HCV and HBsAg than among the subjects who were positive for anti-HCV alone (p < 0.003) or HBsAg alone (p < 0.001); the 5-year cumulative incidence of HCC in the 3 groups was 12%, 2.6% and 0.9%, respectively, and the 9-year cumulative incidence of HCC was 12.0%, 3.0% and 2.0%, respectively. Among the 150,379 subjects who were seronegative for both anti-HCV and HBsAg (81,694 males, 68,685 females; mean age at blood donation, 48.6 years), 27 developed HCC at a mean observation period of 97 months.

Compared with the subjects who were seronegative for both anti-HCV and HBsAg, the age-and-sex-adjusted rate ratios for developing HCC among the subjects positive for anti-HCV alone, subjects positive for HBsAg alone and subjects positive for both anti-HCV and HBsAg were 126, 102 and 572, respectively (Table IV). The synergy index was 2.53, which was significantly higher than unity (95% CI = 1.38–3.68). When the serum ALT and cholesterol levels at blood donation were included as additional independent variables, the rate ratios for subjects positive for anti-HCV alone and for subjects positive for both anti-HCV and HBsAg decreased to 36 and 161, respectively. In the same model, the rate ratio of HCC among subjects positive for HBsAg alone was 74 (Table IV).

Table IV. Independent and Interactive Effects of Hepatitis C Virus and Hepatitis B Virus Infections on the Development of Hepatocellular Carcinoma in a Cohort of 154,849 Voluntary Blood Donors in Osaka, Japan
Anti-HCVHBsAgNumber of subjectsHCCControlled variables1
nAnnual incidence rate (10−5)2Sex, age (95% CI)Sex, age, ALT level (95% CI)Sex, age, ALT level, cholesterol level (95% CI)
  • Anti-HCV (−): negative for second-generation passive hemagglutination test (25 >) at the donor screening; anti-HCV (+): 212 ≤ by second-generation passive hemagglutination test at the donor screening; HBsAg (+), (−): positivity of a reverse passive hemagglutination assay at the donor screening. Synergy index = (572 − 1)/{(126 − 1) + (102 − 1)} = 2.53 (95% confidence interval: 1.38–3.68).

  • 1

    Cox proportional hazard analysis.

  • 2

    Per 105 person-years.

150,379272.21.001.001.00
+1,92752334126 (79–202)52 (31–88)36 (21–62)
+2,51945203102 (63–165)85 (52–138)74 (45–121)
++2531,677572 (173–1,887)270 (80–914)161 (46–557)

DISCUSSION

Although numerous studies have reported the risk of HCC associated with chronic HCV infection, few have presented the absolute risks of HCC among apparently healthy HCV-positive persons, who comprise the majority of HCV carriers, in a large-scale prospective study. Our study showed that the cumulative risk of HCC among anti-HCV-positive, HBsAg-negative voluntary blood donors between the ages of 40 and 74 years reached 21.6% among males and 8.7% among females. While the incidence of HCC was low in the 40–49 year group, it increased with advancing age in both genders. The relatively higher risk of HCC among our subjects compared with that among HCV carriers in reported studies in the United States18, 22 was possibly attributed in part to the better life expectancy of our voluntary blood donors; a better life expectancy would result from lower competing mortalities. Recent reports of cohort studies on Japanese patients with chronic hepatitis C revealed that they had a similar or lower liver-unrelated mortality than the general population of Japan.29, 30

Among the anti-HCV-positive, HBsAg-negative individuals with a normal serum ALT level at enrollment, who comprised 2/3 of the anti-HCV-positive, HBsAg-negative voluntary blood donors, the cumulative incidence of HCC was low. Of the 10 cases (0.76%) with a normal ALT at enrollment who developed HCC, 6 (60%) developed HCC beyond 6 years after enrollment, suggesting that persons with incidentally detected HCV and normal ALT have a potential risk for developing HCC, although the magnitude of the risk is limited. Recent cohort analyses of asymptomatic HCV-positive blood donors who subsequently developed HCC in Taiwan31 and Japan32 may support our notion. In contrast, the subjects with an abnormal serum ALT level, who comprised approximately 1/3 of the anti-HCV-positive individuals in the present study (Table I), had a considerably high incidence rate of HCC that was close to the incidence rate of HCC among patients with histologically determined chronic hepatitis C in tertiary-care treatment facilities formerly reported in Japan.33, 34, 35, 36 This suggests that approximately 1/3 of the community-based HCV-infected persons have a potential risk of developing HCC, which is nearly the same as that among patients with clinically diagnosed chronic hepatitis C in Japan. The effects of these demographic factors and the ALT level on the development of HCC among incidentally detected anti-HCV-positive persons by blood donation indicate the importance of a community-based anti-HCV screening system and provide information on which subpopulations in Japan should be targeted for anti-HCV screening.

On multivariate analysis of the anti-HCV-positive, HBsAg-negative persons, the risk for HCC was significantly higher among males than among females. Differences in drinking and smoking habits between men and women in Japan might have partially influenced this result. Differences in endogenous sex hormones37 may be a factor that caused the difference in the risk for HCC between males and females in this study. An elevated serum ALT level at enrollment was positively associated with the risk of HCC. Any misclassification of the status of liver activity as assessed by the ALT level would likely have been random and would have led us to underestimate any true association in this study. A low serum cholesterol level at enrollment was a risk factor for HCC. It is clear that the association between the serum cholesterol level and the risk of HCC was not affected by the process of selecting the study subjects by a self-administered questionnaire or by donor screening. The serum cholesterol level among patients with chronic hepatitis C was an independent predictor of fibrosis.38 Thus, our subjects with a low serum cholesterol level at enrollment may have had silent hepatic fibrosis evolving to HCC.

Whether coinfection of HBV and HCV is associated with a higher risk of developing HCC than each infection alone is controversial. In one metaanalysis of 21 case-control studies, the synergism was superadditive, with summary odds ratios of 165 for coinfection, 22.5 for infection with HBV alone and 17.3 for infection with HCV alone.39 In contrast, the results of a recent prospective study showed that the interaction between HBV and HCV did not have a significant effect on the development of HCC.21 The present study demonstrated that the interaction between HCV and HBV infections in the development of HCC was superadditive. Regarding the biologic mechanisms of carcinogenesis by these viruses, this finding indicated that chronic HBV infection and HCV infection likely act through common as well as different pathways in the carcinogenic process. The common pathway might be chronic necroinflammatory hepatic change, which in turn acts as a promoter in the multistep process of carcinogenesis. HBV DNA integrates into chromosomal DNA40 and is believed to contribute to the genesis of HCC.41 We showed that the sex-and-age-adjusted rate ratio among subjects who were anti-HCV-positive alone decreased considerably when additionally adjusted for ALT and cholesterol levels (from 126 to 36), whereas the rate ratio among individuals who were HBsAg-positive alone declined slightly in the same analysis (from 102 to 74). This finding supports the existence of a biologic mechanism in which HBV acts in different carcinogenic pathways, as well as in the necroinflammatory process it commonly shares with HCV.

Certain limitations of this study should be considered. We did not confirm the presence of HCV infection by the HCV RNA test. Therefore, some cases may have been false positives. Such misclassification together with overestimation of the number of person-years due to passive surveillance via linkage with the Osaka Cancer Registry would underestimate the absolute risk of developing HCC. The prevalence of anti-HCV among all of the study subjects (1.25%; 1,927/154,850) was somewhat lower than that in a previous study on Japanese blood donors who donated blood in the early 1990s.42 The gap was possibly due to self-selection under the distribution of anti-HCV test in hospitals and clinics from 1992, when the second-generation anti-HCV test became available in Japan. If the self-selection lowered the chance of blood donation by donors with asymptomatic chronic hepatitis C, the cumulative risk of HCC in the anti-HCV-positive donors calculated here would be lower than that among asymptomatic HCV carriers in our community. In Japan, interferon therapy has been available for chronic active hepatitis C patients since the early 1990s and there is evidence indicating that it reduces the incidence of HCC among patients who biochemically respond to it.33, 34, 35, 36 Unfortunately, we could not obtain information as to whether the anti-HCV-positive, HBsAg-negative subjects received any treatment after their enrollment. The absolute risk of developing HCC obtained in this study was thus likely lower than that among HCV-infected persons who do not receive any treatment during their lifetime. Lifestyle factors such as heavy alcohol drinking or smoking43 and virologic factors were not taken into account in this study, while our previous data showed that the HCV genotype in 70% of HCV-positive blood donors in Osaka was type 1b.44

Furthermore, no historical information on how and when the subjects in this study first became infected with HCV was available. The duration of HCV infection was an important factor in determining the outcome of chronic hepatitis C. The time point at which HCV became endemic in Japan was estimated to be between the 1950s and 1970s,5 and the incidence of HCV infection among blood donors in Osaka in the 1990s was very low.24 Based on this circumstance, we speculate that the majority of the anti-HCV-positive subjects in the present study have been infected with HCV for more than 2 decades, and the duration of HCV infection may have been appropriate to assess the risk of HCC in this study.

Our study demonstrated the absolute risk of developing HCC among persons with incidentally detected HCV by using data on voluntary blood donors. The risk of developing HCC was highly dependent on sex, age, serum transaminase level and cholesterol level at blood donation. Coinfection with HBV and HCV is considered to carry a superadditive risk for HCC. These findings would increase public awareness of the necessity of a better anti-HCV screening system in some countries where HCV infection is prevalent. In Japan, a nationwide community-based anti-HCV screening system targeting the age group of 40–70 years is being developed by local municipal governments since 2002.45 The effectiveness of this screening system will be evaluated in the future.

Acknowledgements

The authors thank Ms. Yasue Kotani and the laboratory staff of the Osaka Red Cross Blood Center for their technical assistance.

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