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Anti-hepatitis C virus antibody status and outcomes in serologic assays in blood donors

1 Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Kerman Regional Blood Transfusion Center, Kerman, Iran

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Date of Submission14-Oct-2021
Date of Decision16-Oct-2021
Date of Acceptance29-May-2022
Date of Web Publication12-Dec-2022


BACKGROUND: Hepatitis C virus (HCV) is a major issue in the field of blood transfusion safety and blood components. Serological tests are used to detect anti-HCV antibodies in blood donors. This study was performed to evaluate the status of anti-HCV antibodies in blood donors and serological assays.
SUBJECTS AND METHODS: Data related to the results of screening and confirmation tests of 427,203 blood donors were examined over a 6-year period.
RESULTS: The results showed the highest frequency of positive ELISA results 329 (% 0.39) among first-time blood donors. Out of 901 samples with reactive/positive ELISA result, 104 (11.5%) samples had positive or IND confirmation test results which were belong to donors with a history of healthy blood donation. Antibodies against the NS3 (23%) and core (16.9%) antigens had the highest frequency among blood donors with positive/Reactive ELISA, respectively. The core antigen was the highest immunoreactive protein. A median correlation was found between OD in the ELISA test and the antibody profile in the RIBA test (r = 0.62, P = 0.0001).
CONCLUSION: The seroconversion in healthy blood donors is worrying in relation to the status of the disease in the community and its high incidence. Using Core and NS3 antigens assay can be helpful in screening and diagnosis or antigen detection might be the first next step following a Positive/Reactive antibody test. The use of serological algorithm in blood transfusion centers is questionable and our research has challenged the theory that blood donors with a history of blood donation are healthier in transmitting viral infections.

Keywords: Blood donor, hepatitis C virus antibody, hepatitis C, serologic assay

How to cite this URL:
Mohsenizadeh M, Bamdad T, Ghaziizadeh M. Anti-hepatitis C virus antibody status and outcomes in serologic assays in blood donors. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Jan 28]. Available from:

   Introduction Top

Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus and also classified within the Flaviviridae family, the Hepacivirus genus that causes acute and chronic hepatitis.[1],[2] HCV is a blood-borne virus that can be transmitted through sexual contact, contact with blood and contaminated blood products, organ transplants, and vertical transmission.[3]

Of all infected people, approximately 20%–40% is in spontaneous clearance and in 60%–80% the infection developed to a chronic condition.[4] In most cases, infected patients are asymptomatic and only 20%–30% aware of their disease.[5] The prevalence of anti-HCV antibodies in the global population is estimated at 1.6% or approximately 115 million people.[6]

HCV contains structural and nonstructural proteins that include core, transmembrane glycoprotein's E1, E2 and NS2, NS3, NS4A, NS4B, NS5A, NS5B, respectively.[7] There is no complete and detailed information about the immunogenicity of these proteins in infected people.[1] Although laboratory methods for detecting anti-HCV antibodies in the serum samples of people have made considerable progress, little is known about the humoral immune response to HCV antigens.[7]

Third-generation ELISA kits containing synthetic peptides/antigens include Core, NS3, NS4, and NS5 are used to screen for the detection of antibodies against HCV-antigens.[8],[9] Positive/Reactive ELISA results in individuals are evaluated by recombinant immunoblot assay (RIBA) as a confirmatory test in relation to the presence or absence of anti-HCV antibodies.[10] The Blood Transfusion Organization of Iran monitors the status of hepatitis C infection in blood donors by screening for anti-HCV antibodies and RIBA to increase the positive predictive value of positive anti-HCV assay. In general, enzyme immunoassays (EIAs) and RIBA tests are used to screening and confirmation of anti-HCV antibodies, respectively.[10]

The World Health Organization plans to eliminate hepatitis C by 2030.[11] There is a serious concern about the increasing prevalence of hepatitis C infection and the identification of patients in countries. In this study, the amount of total anti-HCV antibodies in blood donors is measured by the indirect ELISA technique and then the type of specific antibody is determined by the RIBA test. The prevalence and status of antibodies in blood donors and the relationship between these antibodies and blood donation history, blood group, and age are analyzed.

   Subjects and Methods Top

This analytical, cross-sectional study was performed in Tarbiat Modares University, School of Medicine, Department of Virology, and in collaboration with Kerman Blood Transfusion Organization (KBTO).

Tarbiat Modares University approved the study and the ethics approval number was IR. MODARES. REC.1399.086. Inclusion/exclusion criteria, blood donors, aged 18–65 years who have donated and agreed by signing the informed consent form. Exclusion criteria were those who did not meet the inclusion criteria and/or refused to participate. Serum specimens were collected from 427,203 volunteer whole blood donors in the KBTO. Based on the algorithm used in this center, the blood donors screened for anti-HCV antibodies by ELISA assay, the specimens of Reactive ELISA were enrolled in the study. Reactive ELISA specimens were confirmed by semi-quantitative immunoblot assay (RIBA) for use as a more specific supplemental test on specimens.

ELISA test

Total antibodies against HCV antigens (Core, NS3, NS4, and NS5) were measured by 3rd generation ELISA (Monolisa Anti-HCV plus v3 BioRad, EIAgen HCV Ab v4 Adaltis) which is an indirect qualitative EIA with Tecan micro ELISA analyzer, Switzerland and DAVINCI micro ELISA analyzer, bioMerieux, France automation systems. In this algorithm, antibody detection against HCV was first performed by the ELISA method.

General test includes wells coated with virus antigens that complex by adding antibody-containing serum. After incubation, washing and addition of the labeled antibody with the enzyme are performed. Then, after incubation and washing, substrates one and two are added. The dye is formed and the reaction is stopped by sulfuric acid. The results are interpreted after reading the light absorption (OD) by the reader in a period of 30 min with a wavelength of 450 nm. The OD (optical density) values of the samples are considered positive or reactive in cases equal to or greater than the cut-off and negative or inactive results are also considered for light absorption values less than cutoff. Based on the defined algorithm, reactive ELISA cases were evaluated with Supplementary test (RIBA) to identify the type of hepatitis C specific antibody.

Recombinant immunoblot assay

The type of antibody was confirmed by semi-quantitative immunoblot assay (RIBA), using INNO-LIA™HCV Score supplied by INNOGENETICS, Belgium, the MP Diagnostics HCV BLOT 3.0. In this Supplementary test, the results are interpreted based on the brochure of the kit, includes structural and non-structural antigens from HCV, synthetic peptides of the HCV antigens (Core, NS3, NS4, and NS5) are fixed on the strip and has a diagram of the antigens and controls coated on strip. The 3 + intensity is the anti-IgG band and the 1 + intensity band is the IgG control band, these should be visible on all strips. Antibodies in patients' serum bind to these antigens, the intensity of any reactive band is compared to these two bands for reference. Comparison with these bands is performed to assign a reactivity rating to each antigen on the strip. HCV Score test was rated from 1 + to 4 + according to the intensity of the reaction on the antigen line. The interpretation based on the brochure give negative, indeterminate and Positive results.

Statistical analysis

The statistical analysis was performed by IBM SPSS Statistics for Windows, Version 21.0. (Armonk, NY: IBM Corp). The prevalence of anti-HCV antibodies is determined as a percentage of total blood donation. The age and the profile of antigens in RIBA results were calculated with the Pearson correlation coefficient. P < 0.05 (typically ≤ 0.05) was considered a significant level and P < 0.01 was considered a very high level of significance.

   Results Top

Study population

In this survey, we examined a total of 427,203 donors for antibodies against HCV antigens, from January 1, 2014, to January 1, 2020. Among these donors, 410969 (%96.2) were male and 16234 (3.8%) were female. All donations were volunteers. Respectively, 189678 (44.4%), 155074 (36.3%), and 82451 (19.3%) had regular, lapsed, and first-time blood donation. Out of 427203 whole blood donations, 901 (%0.21) ELISA positive/reactive cases were identified, 329 (36.5%) for the first time, 210 (23.3%) were lapsed, and 362 (40.2%) were regular blood donations. The highest frequency of anti-HCV antibodies with ELISA technique (% 0.39) was observed among first-time blood donors.

Anti-hepatitis C virus antibodies to structural (core) and nonstructural (NS3, NS4, and NS5) proteins

The serum sample of the total 901 ELISA positive/reactive cases was confirmed by confirmatory test (RIBA), 620 (68.8%) were negative and considered false-positive ELISA cases, Specimens from 157 (17.4%) blood donors were RIBA positive and 124 (13.8%) were indeterminate, Specific antibodies against HCV were detected in 104 samples that were associated with donors with a healthy donation history, first-time blood donors had the highest number of RIBA positive 131 (14.5%); [Table 1].
Table 1: Confirmation test results

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The profile of HCV antibodies in blood donors with positive/reactive ELISA test results is shown in [Table 2]. Antibodies against the NS3 (23%) and Core (16.9%) antigens had the highest frequency among blood donors with positive ELISA test results, respectively. The highest intensity of the reactive band was observed in association with core antibodies. The frequency of blood groups in blood donors with positive antibody profile is shown in [Table 3].
Table 2: The profile of anti-hepatitis C virus antibodies in positive/reactive ELISA test results

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Table 3: Blood groups frequency in blood donors with positive antibody profile and the total population of blood donors

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The effect of age on the results of antibody profiles in RIBA was investigated, which showed little/no correlation between the effect of age on the results of antibody profiles [P = 0.001, r = 0.17; [Table 4]] and also, the correlation between OD in ELISA test and the results of antibody profiles in RIBA was investigated [P = 0.0001, r = 0.62; [Table 4]].
Table 4: The effect of age and optical density in ELISA test on the results of antibody profiles in recombinant immunoblot assay

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   Discussion Top

During hepatitis C infection, the appearance of biomarkers is included, respectively: Viral RNA, antigenemia and anti-HCV antibodies. Anti-HCV IgMs are not a reliable marker in the diagnosis of acute infection, and therefore, is not used in serological diagnostic tests, therefore, indirect serological tests do not distinguish the current from the past infection.[10],[12] Total IgG antibodies are usually measured by the EIAs technique.[13] Conserved antigens that prepared from the HCV core, NS3, NS4, and NS5 genomic regions are used in the third generation ELISA assay to detect anti-HCV antibodies.[10]

In this study, with initial screening for Anti-HCV antibodies by ELISA, 901 (%0.21) whole blood donors were ELISA positive/reactive. A reactive anti-HCV test result can be due to a false-positive reaction/past infection/current active infection.[14] The highest frequency of positive ELISA results (% 0.39) was observed among first-time blood donors, this is less than the results obtained (2.49%) in the general population in other provinces of Iran.[15] This discrepancy may be due to a medical interview and examination performed at the time of acceptance of the blood donor to promote the health of the donated blood. Different results (52%) were obtained with different anti-HCV assays and a significant difference (58% up to 80%) was observed in the inter-assay concordance in the ELISA methods.[16] Considering the results of the initial ELISA test in first-time donors, it should be noted that the prevalence of hepatitis C infection in the general population of Kerman province needs to be seriously investigated.

580 (64.4%) samples of reactive anti-HCV antibody belong to donors with a history of healthy blood donation and 104 (11.5%) samples from these donors have positive or IND confirmation test results [Table 1]. The high rate of seropositive serum cases among these donors can be due to the doctor's overconfidence during the pre-donation interview with the donor, concealment of risk factors by the donor and donation of blood during the window period (serological window period) and also high incidence of the disease in the General population or due to the inaccuracy and sensitivity of this algorithm used in Iran.[17] The serological window in hepatitis C infection varies and also the detectable antibodies vary from patient to patient, the window period has been reported in several studies from a few days to 9 months[9],[18],[19]. There are two theories here to justify test results: Existing diagnostic tests are unable to detect low-level immune responses, or multiple test results are due to cross-reaction to other pathogens or peptides.[20] Using a more sensitive threshold in the serological test signal to cut-off ratio can lead to the identification of people with occult HCV infection.[21] Seronegative HCV infection and occult HCV infection should be investigated in blood donors.[19] Therefore, if we use a serological algorithm, we must also consider seroreversions and/or seroconversions in blood donors.

The confirmation test was performed on 901 samples with reactive/positive ELISA results, of these, we identified specific antibodies against virus antigens in 283 (31.4%) samples. The absence of anti-HCV antibodies can be due to acute infection, immunocompromised patients, agammaglobulinemia, and occult HCV infection and in rare cases it has also seen in blood donors.[10],[19] In low endemic areas and low-prevalence populations, false-positive HCV antibodies (anti-HCV) are more than 70% and the positive predictive value of Anti-HCV assays decreases (68%), respectively.[21],[22] The immune response to HCV peptides varies in different races and regions.[23]

By performing the confirmatory test to identify specific antibodies to virus antigens, we had different antibody profiles. The highest frequency of detected antibodies was related to NS3 antigens. Antibody to NS3 may be associated with viral persistence, the antibody response to NS3 is higher in people with viral persistence than in those with apparent viral clearance from blood.[1] The humoral immune response begins with the production of antibodies against epitopes of the nonstructural NS3 and viral capsid proteins, as the infection persists, antibodies against NS4 epitopes can be detected.[13] Decrease antibody to NS3 is significantly associated with clearance of the virus from the blood and also NS3 antibody can be considered as a marker of chronicity in blood donors.[1] Multiple reports among patients who are seronegative/HCV-unexposed/uninfected Shown a history of immune response to viral peptides including NS3, this is may be due to cross-reaction with unrelated pathogens include: Influenza virus neuraminidase, human herpesvirus type 1 (HHV-1), influenza A virus, Another study found that 20% of people who do not have a history of HCV have a memory of immune response to HCV.[20] The identification of antibody to NS3 in regular and lapsed donors who have a history of negative test results in several cases of blood donation may be due to the reactivation of the virus in blood donors with hepatitis C or the Quality of testing process at different times which leads to IND results in the RIBA test. Studies in Patients with occult HCV have shown that they can be seronegative or had low OD in ELISA test but had IND and seroreactivity to NS3 protein in the supplemental immunoblot assay.[21]

The highest color intensity associated with the core antigen bond was observed in the specific position on the nitrocellulose strip [Table 2]. Therefore, the core antigen was the highest immunoreactive protein. The most immunoreactive antibody in patients with acute and chronic hepatitis C infection belongs to the core antigen.[12] In our study, the highest immunogenicity of HCV proteins was Core, NS3, NS4, or NS5, respectively. The results of other studies confirm this findings.[7] Other studies have shown that HCV-NS3 antigen and HCV-Core antigen are the acceptable markers for screening and diagnosis.[24]

The highest prevalence of anti-HCV antibodies was observed in blood groups A+ (0.07%) and O+ (0.07%) [Table 3]. There was no significant relationship between the age of blood donors and the results of antibody profiles [Table 3]. Most blood donors are under the age of 45, and this connection cannot be considered logical, other studies have shown this.[17],[25]

A median correlation was found between OD in the ELISA test and the antibody profile in the RIBA test. The amount of OD cannot be a good criterion for predicting the results in this algorithm. This data is an alarm in relation to the role of using serological algorithms in blood donors. This algorithm has many ambiguities that need to be investigated. Blood transfusion centers should upgrade this algorithm with molecular tests, liver tests, and testing for virus antigens.

   Conclusions Top

Our data in this study showed that, a number of blood donors with a history of healthy blood donation became seropositive during the study period, which highlights the importance of seroconversion in this algorithm. Due to the elimination of high-risk donors or blood donors with risk factors in medical interviews, this issue is worrying in relation to the status of the disease in the community and its high incidence. The core antigen was the highest immunoreactive protein and the highest frequency of detected antibodies was related to NS3 antigens, using core antigen assay and NS3 antigen assay can be helpful in screening and diagnosis or HCV antigen detection might be the first next step following a positive antibody test. The use of serological algorithms to identify patients or screening requires detailed studies of various dimensions that must be considered. Therefore, in order to cover the gaps of this algorithm, the use of the liver function tests, antigen, and molecular identification should be developed. With these results, the use of serological algorithms in blood transfusion centers is questionable and our research has challenged the theory that blood donors with a history of blood donation are healthier in transmitting viral infections. This algorithm in Iran should be reviewed as soon as possible.


The authors would like to acknowledge KBTO.

Financial support and sponsorship

This study was financially supported by under the grant of Tarbiat Modares University.

Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Taravat Bamdad,
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, P.O.Box: 14115-111, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.AJTS_156_21


  [Table 1], [Table 2], [Table 3], [Table 4]


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