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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 2  |  Page : 205-210

Performance evaluation of blood donor screening assays for serological detection of Hepatitis B surface antigen and antibodies to Hepatitis C virus


1 Department of Transfusion Medicine, Bharati Vidyapeeth Medical College Hospital and Research Centre, Pune, Maharashtra, India
2 Department of Transfusion Medicine, Institute of Liver and Biliary Sciences, New Delhi, India

Date of Submission11-Aug-2021
Date of Decision20-Oct-2021
Date of Acceptance21-Oct-2021
Date of Web Publication30-Nov-2021

Correspondence Address:
Dr. Meenu Bajpai
Department of Transfusion Medicine, Bharati Vidyapeeth Medical College Hospital and Research Centre, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/gjtm.gjtm_79_21

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  Abstract 


Background and Objectives: Screening for transfusion transmissible infections in donated blood can be done by either rapid diagnostic tests (RDTs) or enzyme-linked immunosorbent assay (ELISA) or any other available sensitive immunoassay such as chemiluminescence immunoassay (CLIA). The aim of this assay was to evaluate performance of two commercially available RDTs and CLIA against ELISA for serological screening of hepatitis B surface antigen (HBsAg) and anti hepatitis C virus (HCV). In addition, we also tested the seropositive samples by RDTs, CLIA, and/or ELISA by molecular assays (transcription-mediated amplification, transcription-mediated amplification, and quantitative polymerase chain reaction). Methods: In this prospective study, 1000 consecutive blood donors were screened from September 2017 to March 2018. All blood samples collected during the study period were tested by RDTs, CLIA, and ELISA, and the results obtained were compared. Results: On considering ELISA as a reference standard, low analytical sensitivity was noted for both RDTs (64.29%) and CLIA (71.43%) for HBsAg detection. Similar results were also noted for both RDTs (25%) and CLIA (75%). The positive predictive value of CLIA was found to be lower (HBsAg 31.25%; anti-HCV 50%) as compared to RDTs (HBsAg 90%–100%; anti-HCV 100%). High false positivity was noted with CLIA, while RDTs missed seropositive samples. The viral load for HBsAg and anti-HCV seropositive samples ranged from 29.7 IU/ml to 3.4 × 104 IU/ml and 7.56 × 106 IU/ml, respectively. Conclusions: Performance of CLIA as screening assay was better compared to RDTs. CLIA seems to be a suitable screening assay for emergency situations and predonation apheresis donor screening. RDTs may be used as supplemental assay prior to donor notification.

Keywords: Blood donor, performance, screening assay, sensitivity, transfusion-transmitted infections


How to cite this article:
Bajpai M, Kakkar B, Gupta E, Kumar G. Performance evaluation of blood donor screening assays for serological detection of Hepatitis B surface antigen and antibodies to Hepatitis C virus. Glob J Transfus Med 2021;6:205-10

How to cite this URL:
Bajpai M, Kakkar B, Gupta E, Kumar G. Performance evaluation of blood donor screening assays for serological detection of Hepatitis B surface antigen and antibodies to Hepatitis C virus. Glob J Transfus Med [serial online] 2021 [cited 2022 Jan 18];6:205-10. Available from: https://www.gjtmonline.com/text.asp?2021/6/2/205/331625




  Introduction Top


It is the prime responsibility of blood transfusion services (BTS) to provide safe blood.[1] In India, it is mandatory to test the donated blood human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV). Apart from these tests, the donated blood is also screened for malaria and syphilis as mandated by the Drugs and Cosmetics Act, 1940 and Rules, 1945.[2],[3]

Transfusion-transmissible HIV, hepatitis B virus (HBV), and HCV have always been of great concern due to their prolonged viremia and chronic carrier state.[4] The risk of posttransfusion HCV/HBV is higher (12.5%) as compared to posttransfusion HIV (5%–10%).[4] Globally, viral hepatitis has also emerged as a leading cause of death in 2015, overtaking deaths due to HIV/AIDS and tuberculosis.[5] Over the last decade, the focus has shifted from HIV to HBV/HCV.

The various screening assays available are rapid diagnostic tests (RDTs), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA). Additional screening assays such as nucleic acid testing are also available and are in use as an additional layer of safety.[1],[6] The type of screening assays used for TTI testing varies from one transfusion service to another depending on the number of blood units collected and availability of resources. ELISA is the most widely used serological assay for blood screening. In BTS with a limited number of donations and resource constraints, the donated blood is screened by RDTs.[6] Central Drugs Standard Control Organization (CDSCO-India) recommendations issued in 2013 stated that any in vitro diagnostic devices approved for diagnostic purposes can be utilized for screening of donated blood; as the criteria such as sensitivity and specificity are the same.[7],[8] Thereafter, CLIA emerged as a new entrant for the screening of donated blood on an automated platform in India.

Aims and objectives

A good screening assay is an important aspect of blood safety. Therefore, we aimed to evaluate the performance of two commercially available RDTs and CLIA against ELISA (gold standard) for serological screening for HBV and HCV in the blood bank scenario where the tested population is apparently healthy and the prevalence of these infections is low. Our secondary objective was to test the seropositive samples by RDTs, CLIA, and/or ELISA by transcription-mediated amplification (TMA) and quantitative polymerase chain reaction (qPCR).


  Materials and Methods Top


This retrospective cross-sectional study was conducted from September 2017 to March 2018 in the department of transfusion medicine at a tertiary care hospital.

ETHICS – This study was done after obtaining the ethical clearance from the institutional ethics committee. A total of 1000 consecutive whole blood donors eligible to donate and consenting (written infromed consent) to participate were included in the study. Haemolyzed and lipemic blood samples were excluded.

Blood samples were collected in 2 ethylene diamine tetraacetic acid vials, which were then centrifuged and the plasma was separated into 2 aliquots of 3 ml each (aliquot 1: testing by serological assays; aliquot 2: testing by molecular assay). All blood samples collected during the study period were tested for HBsAg and anti-HCV by 2 RDTs each, CLIA and ELISA [Table 1]. All testing was done prior to the release of blood into the bloodstock inventory. In case of nonreactive results in all the serological assays, blood components were released into the inventory, whereas in case of reactive or discordant results (non-agreement between the results obtained by RDTs, CLIA, or ELISA), they were discarded, following which samples were tested by molecular assay based on TMA (qualitative assay) and viral load was estimated using qPCR [Table 1]. [Figure 1] summarizes the algorithm used in the present study.
Table 1: Specifications of serological and molecular assays used

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Figure 1: Serological testing algorithm for hepatitis B surface antigen and anti-hepatitis C virus among blood donors

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Statistical analysis

The collected data were entered into an Excel spreadsheet. A comparative performance assessment of the following parameters was performed: sensitivity, specificity, positive predictive value (PPV), negative predictive value, and Youden's weighted index using standard formulae.[9] Cohen's kappa test was performed to determine the agreement between the molecular assays (TMA and qPCR). P <0.05 was considered to be statistically significant. Statistical analysis was performed using IBM SPSS statistical software (version 20, SPSS Inc., Chicago, IL, USA).


  Results Top


The study included a total of 1000 (97.1% males; 2.9% females) consecutive whole blood donors with a median age of 29 years (18–60). All 1000 samples (same donor) were tested by RDTs, CLIA, and ELISA. A total of 32 and 6 donors samples were reactive for HbsAg and anti-HCV, respectively by RDTs, CLIA, or ELISA. None of these samples were reactive for anti-HIV I and II, malaria, or syphilis. Seroprevalence of HbsAg and anti-HCV was 1.4% and 0.3%, respectively.

Comparison of rapid diagnostic tests and chemiluminescence immunoassay against enzyme-linked immunosorbent assay

[Table 2] and [Table 3] summarize the seroreactive status of samples tested by RDTs and CLIA against ELISA. The analytical sensitivity of RDTs for HBsAg detection was 64.29% with a specificity of 99.9%–100%, while CLIA showed low sensitivity and comparable specificity to RDTs, 71.43% and 97.77%, respectively. PPV of CLIA was found to be lower (31.25%) in comparison to RDTs [90%–100%; [Table 4]].
Table 2: Serostatus of hepatitis B surface antigen samples (n=1000) tested by rapid diagnostic tests and chemiluminescence immunoassay against enzyme-linked immunosorbent assay (gold standard)

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Table 3: Serostatus of anti-hepatitis C virus samples (n=1000) tested by rapid diagnostic tests and chemiluminescence immunoassay against enzyme-linked immunosorbent assay (gold standard)

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Table 4: Performance of rapid diagnostic tests and chemiluminescence immunoassay against enzyme-linked immunosorbent assay

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The analytical sensitivity of RDTs for anti-HCV detection was 25% with a specificity of 100%, while CLIA showed low sensitivity and comparable specificity to RDTs, 75% and 99.7%, respectively. PPV of CLIA was found to be lower (50%) in comparison to RDTs (100%). Thus, high false positivitywas noted with CLIA, while RDTs missed seropositive samples for both HBsAg and anti-HCV [Table 4].

Testing of seropositive samples by rapid diagnostic tests, chemiluminescence immunoassay, and/or enzyme-linked immunosorbent assay by transcription-mediated amplification and quantitative polymerase chain reaction

qPCR was performed on 38 samples seropositivge by RDTs, CLIA, or ELISA (32 HBsAg; 6 anti-HCV) to compare the results with TMA and determine the viral load. Of the 32 HBsAg seropositive samples, HBV DNA was detected in 7 (21.9%) samples by TMA and in 10 (31.3%) samples by qPCR. The viral load for these samples ranged from 29.7 IU/ml to 3.4 × 104 IU/ml. The viral loads of the three samples missed by TMA were 29.7 IU/ml, 79.2 IU/ml, and 7.08 × 102 IU/ml, respectively. Concordance between TMA and qPCR for HBV DNA detection was 90.6% with moderate agreement (κ =0.762; P ≤ 0.001). The 6 anti-HCV seropositive samples tested by both TMA and qPCR showed HCV RNA in one sample (same sample) only. The viral load of this sample was 7.56 × 106 IU/ml. Concordance between TMA and qPCR for HCV RNA detection was 100% with perfect agreement (κ =1.000; P = 0.014).


  Discussion Top


Varied types of screening assays are used for screening TTIs in blood donors worldwide as there is no consensus regarding the optimum assay for blood donor screening. The desirable qualities on which assay selection depend are assay performance, workload, cost, technical skill, and turnaround time (TAT). An ideal assay would be able to differentiate between those who have or those who do not have an infection and be cost-effective and non-invasive.[10] When we consider all practical issues, an optimal screening assay would be the one with high sensitivity (~100%) with an acceptable specificity to detect all true positive samples; however, some amount of wastage due to false positivity would be acceptable.

When ELISA was considered as a reference assay, RDTs showed low sensitivity (HBsAg 64.29%; anti-HCV 25%) with a high specificity (HBsAg 99.9%–100%; anti-HCV 100%) in this scenario. Similar results have been reported Dogbe and Arthur, Bjoerkvoll et al., Grijalva et al., Kaur et al., Mutocheluh et al., and Torane and Shastri.[11],[12],[13],[14],[15],[16] RDTs are considered a good screening assay if they demonstrate a high PPV with a low false-negative rate.[17] Although we observed a high PPV for RDTs, the low sensitivity and consequent high false-negative rate indicated their poor performance as screening assays. Even, the World Health Organization does not recommend the use of RDTs as a screening assay as they are designed for the immediate and rapid testing of a small number of samples, mainly for diagnostic purposes.[1] The various reasons for seropositive samples being missed by RDTs are differences in the genotype/subtype, suboptimal design of antigens, low viral load, and mutants.[11],[12],[13],[14],[15],[16] There was one sample which was found to be seronegative for HBsAg by RDTs; however, the same sample was found to be seropositive by CLIA and ELISA. Further testing of this sample by TMA and qPCR revealed the presence of HBV DNA with a low viral load (30.6 IU/ml), thus highlighting that RDTs tend to miss low viral load samples which compromises blood safety.[11],[12]

We also observed that on considering ELISA as a reference standard, CLIA showed low sensitivity (HBsAg 71.43%; anti-HCV 75%) with high specificity (HBsAg 97.77%; anti-HCV 99.7%). PPV of CLIA was also found to be low for both HBsAg (31.25%) and anti-HCV (50%), thus high false positivity was noted. Testing with CLIA resulted in a higher wastage of the resources due to discarding of blood components labeled as seropositive. The various reasons for false-positive results are testing with lipemic samples, cross-reactive antibodies, and interfering substances present in the serum.[11],[12],[13] False-positive results cause psychological stress to the donors when they are notified about the results of screening.[18],[19],[20],[21],[22] In spite of this, higher false-positive results are more acceptable than missing out on true positives and compromising the blood safety.[20]

It is known that ELISA shows high sensitivity and specificity but is not suitable for use in emergency situations such as screening of rare blood group donors and predonation screening of apheresis donors due to its high TAT (3–4 h) as compared to RDTs and CLIA (~30 min).[23] Taking into cognizance the poor performance of RDTs, CLIA seems to be a better screening assay as it is exclusively available on an automated platform with the inherent advantages of automation such as increased sensitivity, high-speed throughput, multi-analyte analysis on a single platform, low consumption of reagents, and high stability of their reagents and conjugates. Apart from these, the additional advantages are a short TAT and random access as compared to automated ELISA, thus making it more suitable for use in emergency situations and predonation apheresis donor screening. However, the disadvantages of CLIA are high number of false positives, cost, technical expertise, and regular equipment maintenance to maintain reproducibility of the assay.[24],[25] Despite the disadvantages of CLIA, it still seems an optimum screening assay.

Another interesting finding in our study was that three donor samples tested for HBsAg (reactive by all the serological assays) were missed by TMA and further testing of these samples by qPCR revealed a low viral load ranging from 29.7 IU/ml, 79.2 IU/ml, and 7.08 × 102 IU/ml, respectively. Nübling et al. have also reported that breakthrough transmission of HIV and HCV due to molecular assay failures is possibly due to low viral load and/or suboptimal amplification efficiency of the molecular assay.[26] Despite having sensitive screening assays available, there is a chance that qualitative molecular assays can miss low viral loads; therefore, provision of zero risk blood still seems a distant dream for BTS. Therefore, there is a need to test the blood donor samples by both serological and molecular assays simultaneously so that the samples missed by one technique may be picked up by the other, thus, improving the overall blood safety from TTIs. Even in this era of molecular screening assays, the importance of serological screening assays cannot be undermined.

The strengths of our study were (i) large sample size, (ii) depicted the actual scenario of testing donated blood, (iii) highlighted the significant differences between observed and manufacturer claimed sensitivities for RDTs, and (iv) it also highlighted the strengths and shortcomings of CLIA as a screening assay. However, our study was not without limitations. First, it was done at a single center. Second, molecular testing could not be performed on all the samples due to resource restrictions. The results for HCV should be interpreted with caution as only one sample was found to be true positive for HCV by both TMA and qPCR.


  Conclusion Top


The performance of CLIA as a screening assay was better as compared to RDTs. ELISA would be ideal in BTS where batch testing of blood donor samples is the norm, whereas CLIA seems to be a suitable screening assay for BTS where we come across emergency situations, centers providing round-the-clock transfusion services and predonation apheresis donor screening due to short TAT and random access. Consideration may be given to use of RDTs as supplemental assay due to its high specificity and PPV prior to donor notification to prevent psychological stress of donors due to false-positive results of CLIA/ELISA. Detection of low viral load samples by molecular screening assays highlights that to ensure blood safety, there is a need to test donor samples with both serological and molecular assays.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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World Health Organization. Screening Donated Blood for Transfusion-transmissible Infections: Recommendations. Geneva: World Health Organization; 2009. Available from: https://www.who.int/bloodsafety/ScreeningTTI.pdf. [Last accessed on 2018 Dec 12].  Back to cited text no. 1
    
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