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Year : 2021  |  Volume : 6  |  Issue : 2  |  Page : 160-165

High yield of minipool NAT in India using a sensitive multiplex assay for blood donor screening

Radhey Shyam Gupta IMA Blood Bank, Bareilly, Uttar Pradesh, India

Date of Submission22-Jan-2021
Date of Decision20-Oct-2021
Date of Acceptance21-Oct-2021
Date of Web Publication30-Nov-2021

Correspondence Address:
Dr. Anju Uppal
Radhey Shyam Gupta IMA Blood Bank, Bareilly, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/gjtm.gjtm_7_21

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Background and Objectives: Nucleic acid amplification testing (nucleic acid testing [NAT]) for blood donor screening is increasingly being adopted in India to reduce the risk of transfusion-transmitted diseases. The high sensitivity of NAT enables testing of a large volume of donations in minipool format; reducing the costs of testing compared to individual donation testing. This study was aimed at evaluating the yield of minipool NAT testing for hepatitis B, hepatitis C, and human immunodeficiency virus (HIV) in blood donors from Uttar Pradesh. Methods: Samples from routine blood donors collected between August 16, 2016, and December 31, 2020, that were seronegative for hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV, syphilis, and malaria were further screened by NAT in pools of six donations (MP6) using the cobas TaqScreen MPX Test, version 2.0 (MPX2) on the cobas s 201 System. Members of reactive pools were tested to identify the reactive donation(s). Viral load testing was performed on randomly selected NAT-reactive samples. Results: Of 172,443 seronegative donors, 463 were NAT reactive: 369 HBV, 89 HCV, 2 HIV, and 3 co-infected with HBV and HCV. The overall NAT yield was 0.27%, with individual virus yield rates of 1/464 for HBV, 1/1874 for HCV, and 1/86,222 for HIV. Viral load testing of 19 HBV NAT-yield donations showed low concentrations in 16 samples and undetectable viral load in three; the two HCV NAT-yield donations tested had high viral loads. Conclusion: Screening of blood donations by minipool NAT using the highly sensitive MPX2 assay identified a high yield of serology negative, NAT-positive donations, including HBV reactive donations with low viral concentrations.

Keywords: Blood donor screening, minipool, nucleic acid testing yield, nucleic acid test, viral load

How to cite this article:
Uppal A, Priya P, Sethi JP. High yield of minipool NAT in India using a sensitive multiplex assay for blood donor screening. Glob J Transfus Med 2021;6:160-5

How to cite this URL:
Uppal A, Priya P, Sethi JP. High yield of minipool NAT in India using a sensitive multiplex assay for blood donor screening. Glob J Transfus Med [serial online] 2021 [cited 2022 Aug 10];6:160-5. Available from: https://www.gjtmonline.com/text.asp?2021/6/2/160/331618

  Introduction Top

Addition of nucleic acid testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) to standard serology screening for blood donations provides increased overall screening sensitivity. The availability of automated molecular screening systems enables blood services to implement molecular screening without the need for special laboratory facilities and minimizes the hands-on time required by staff to perform the testing.

India is a country with a large population, an estimated need for over 12,000,000 donations per year[1] and a high level of infectious agents in many areas.[2],[3],[4] The implementation of molecular screening could significantly reduce the risk of transmission of viral infections through transfusion. There are some published data available to support this,[5],[6] but as technological advances are made and fully automated molecular screening systems become available, the potential benefit of such systems requires more detailed investigation. In this report, our blood center evaluates the incremental yield of minipool NAT compared to serology testing. The data include screening and detection of infectious units that are serology nonreactive and DNA/RNA reactive and quantitation of the viral targets. We examined data to see it supports the benefits of molecular testing in minipool format using a highly sensitive NAT, such as the cobas TaqScreen MPX Test, version 2.0 (MPX2) for use on the cobas s 201.

Aims and objectives

This study examines the need for Minipool NAT, and the need for further investigation of screened reactive donations to understand the status of the infected donor.

  Materials and Methods Top


Samples collected from blood donors of a standalone blood bank in India between August 16, 2016, and December 31, 2020, were tested by routine serology. All donations nonreactive by serology were further tested by NAT. A total of 179,245 donations were collected and underwent serology screening for HBV, HCV, and HIV. Of these: 42.0% (75,285/179,245) were from repeat donors, the remainder from first time donors; 62.4% (111,786/179,245) were from voluntary donors, the remainder from replacement donors; and 97.2% (174,232/179,245) were from male donors.

Serological screening

Serological screening was performed, following routine procedures, for: HBsAg (Monolisa HBsAg Ultra; sensitivity 100%), HCV Ag/Ab (Monolisa HCV Ag/Ab Ultra v2; sensitivity 100%), HIV Ag/Ab (Genscreen Ultra HIV Ag/Ab; sensitivity 100% in confirmed antibody positive samples and 94.6% in antigen positive samples) all from Bio-Rad, Marnes-la-Coquette, France;[7],[8],[9] syphilis (RPR-Carbogen, Tulip Diagnostics, Goa, India); malaria antigen (Advantage Malaria Ag, J Mitra, New Delhi, India) or by standard microscopy. All assays were performed according to the manufacturers' instructions.

Any donation found to be initially screen reactive for any of the serological targets was considered “serology screen positive” and NAT was not performed on these donations.

Molecular screening

NAT was performed using the cobasTaqScreen MPX Test, version 2.0 for use on the cobas s 201 System (MPX2)(Roche Molecular Systems, Branchburg, NJ, USA) including the automated Hamilton MICROLAB® STARlet IVD Pipettor sample pooler. The MPX2 assay detects HIV-1 Group M RNA, HIV-1 Group O RNA, HIV-2 RNA, HCV RNA, and HBV DNA and uses the polymerase chain reaction technology.

NAT was performed on the pools of six samples (MP6) following the manufacturer's instructions for the assay and automated platform.[10]

Reactive pools were resolved by testing the six donations individually to identify the reactive donation(s). The result of the MPX2 test is reported as reactive for a specific target(s), i.e. HBV, HCV, or HIV.

The reported 95% and 50% detection limits for MPX2 are shown in [Table 1].[10],[11]
Table 1: Detection limits for the molecular assays used in this study[10],[11],[12],[13],[14]

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Additional/follow-up testing

Viral load was determined for some of the NAT yield donations. Retained samples from NAT yield donations were randomly selected from the frozen storage and sent for viral load testing as resources allowed. The samples were stored between 10 days and 3 months at -40°C. Testing was performed at another center using the cobas TaqMan HBV test, HCV test v 2.0 and HIV test v 2.0; all three tests were those designated for use with the Roche High Pure System, run on a cobas TaqMan 48 Analyzer (Roche Molecular Systems Inc., Branchburg, USA). Manual nucleic acid extraction was performed using the Roche High Pure System (Roche Molecular Systems Inc., Branchburg, USA). The limits of detection and quantification for the viral load assays are shown in [Table 1].[12],[13],[14]

Donors of serology negative/NAT-reactive donations were asked to return for follow-up testing. Serological follow-up was performed using the same assays that had been used for the initial serology screening.


All procedures in this study were followed in accordance with the standards of the institution's ethics committee on human experimentation and with the Helsinki Declaration of 1975, revised in 2000.


The data were entered in Microsoft Excel. Descriptive statistics were used, and no statistical tests were performed.

  Results Top


Serological screening was reactive on 6186/179,245 donations [Table 2]. No further investigations were performed on these screen-reactive donations.
Table 2: Serology screen reactives in 179,245 donations tested

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A total of 172,443 donations were screened for viral nucleic acids; 6186 donations were excluded due to initial serological screen reactivity. Another 616 donations were excluded for a range of reasons for which the donations were unsuitable for clinical use.

A total of 463 (0.27%) donations, 46 voluntary and 417 replacement donations, were found to be NAT reactive, but three of these were found to be dual infections, identifying a total of 466 infections in the 463 donations. NAT results and yields are presented in [Table 3]. The difference in NAT yield between voluntary and replacement donations was significant, P < 0.0001.
Table 3: Nucleic acid test yield in 172,443 seronegative donations tested

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Additional/follow-up testing

Viral load

Viral load was performed on a small number of samples, i.e., 21/463 (4.54%) NAT yield donations. Of the 21 samples tested, 19 (90.5%) were HBV positive and 2 (9.5%) were HCV positive [Table 4]. The results recorded as < 6 IU/ml were reported as such by the assay, meaning that HBV DNA was detected, but less than 6 IU/mL, the minimum quantitative value reported by the assay.
Table 4: Viral load data

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Follow-up investigations

Although all donors of the NAT yield donations were asked to return for follow-up after 2 months, only 3/463 (0.65%) donors returned. None of the donors for which viral load testing had been performed returned for follow-up.

The follow-up serology results for those donors who did return are presented in [Table 5]. No molecular investigations were performed on these samples.
Table 5: Follow-up serology results

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  Discussion Top

The data presented in this study clearly demonstrate the ability of minipool NAT to generate incremental yield over serology alone. The additional testing, although performed only on a small subset of the NAT yield donations, provides interesting additional data, which may help understand the stage of infection in these serology screen negative donors. Although other published data from India on NAT exist, the data generated by this study and their interpretation, are strengthened by the size of the study, the additional quantitation data available, and although limited, the follow-up serology.

The data show an overall incremental yield, based on initial pool reactivity with subsequent pool resolution, of 0.27% with individual virus yield rates of 1/464 for HBV, 1/1874 for HCV and 1/86,222 for HIV. Not surprisingly, given the high incidence and prevalence of HBV in India,[15],[16] the greatest yield is for HBV. These data are in general agreement with other published Indian blood screening NAT data, a finding that is important as the data in the current study were generated using pooled NAT. This demonstrates the high yield of NAT screening by pooled NAT in India when using an assay with high sensitivity for HBV, such as the cobas TaqScreen MPX Test, v2.0. Of note is that 9/19 (47%) HBV NAT yield donations had viral loads <6 IU/mL. Chandra et al. reported similar results with an earlier version of cobas TaqScreen MPX also performed in MP6.[17] In that study, 35,022 serology-negative donors were screened by NAT, of which 27/108 HBV NAT-reactive donations had low viral loads of <6 IU/mL.

A significant benefit of the current study to the subject of NAT yields in India is the viral load quantitation performed on a subset of the NAT yield samples. Most importantly, the viral load testing confirms that screening with MPX2 in MP6 detects samples with very low HBV viral loads. It is unfortunate that only a small number of the NAT yield samples underwent viral nucleic acid quantitation. The initial study plan was to randomly select a subset of NAT yields on a quarterly basis, but failure in the long-term storage system resulted in the loss of some stored samples. Nonetheless, the 21 samples that were referred for quantitation returned interesting and useful results. Although the assays used for quantitation were less sensitive than the MPX2 screening assay, the presence of viral nucleic acid was identified in the majority of the samples, with HBV DNA at low levels in all of the HBV samples and HCV RNA at high levels in both of the HCV samples. This reflects the differences in the biology of the two viruses and further suggests that the HBV infections are occult hepatitis B infection (OBI) or possibly recent or resolving infection, prior to the appearance of HBsAg or at the point at which HBsAg has declined to below detectable levels and prior to the clearance of circulating HBV DNA. Although limited, the serological follow-up data, the appearance of HBsAg in one of the two HBV follow-ups and the appearance of HCV Ag/Ab in the HCV follow-up, suggest that some of the NAT yields could be due to recent infection, donations being collected in the serological window period. This possibility is supported by the data published by Mishra et al. in 2017, who found that 31/44 HBV, 5/5 HCV, and 5/5 HIV NAT yield donations they identified seroconverted within 6 months. It is quite likely that such data are not necessarily representative of India as a whole. It is likely that in India, like other regions of higher HBV endemicity, the frequency of OBI would be higher than acute infections.[18]_The lack of full definition of the phase of HBV infection in HBV NAT-yield donations detected in India has been highlighted in the work of Doda et al.[19] who looked at the HBV antibody profile of HBsAg negative, HBV DNA positive donations; anti-HBc, anti-HBe and anti-HBs. Of the 18 samples profiled, only 12 had anti-HBc present, the other six had no detectable serological markers. As expected, the data for HCV support the expectation that these NAT yields would all be due to infections in the serological window period. The high viral loads in the HCV NAT yield samples that were quantified are consistent with the rapid doubling time of HCV in early infection.[20] The follow-up of the donors of NAT yield donations, especially HBV yields, would be very useful in trying to properly understand the nature of these donors, both in terms of the threats to the blood supply and how to mitigate them, and in any clinical needs/public health issues associated with these individuals.

Overall, the NAT yields vary between published reports from India, presumably reflecting both variations in donor populations and to some degree assay performance. In 2017, Ghosh and Mishra[21] reviewed the data from published blood bank NAT yield studies, and identified yield ranges for HBV from 1/500 to 1/7000; HCV from 1/1500 to 0/70,000, and HIV from 1/24,000 to 0/70,000. Although wide ranging, these data all show a similar picture, and in agreement with this study, that highest NAT yields are for HBV, and HIV NAT yields are relatively rare. Published data since this study provide broadly similar data.[22] Overall, combined NAT yields of between 1/1000 and 1/4500 are seen, indicating a significant benefit of NAT in helping to minimize the risk of DNA/RNA positive but serology-negative donations entering the blood supply. Interestingly, some of the studies have identified co-infections, all being dual HBV and HCV, as was detected in this study. The overall HIV yield is at least one order of magnitude lower than the HCV yield, indicating a generally low level of HIV in the donor population and/or the effectiveness of serology in identifying donations from HIV infected donors.

The NAT yield in this study is among the highest reported in India even though most other authors who reported NAT yields performed NAT by individual donation testing. We are aware of only one report of a higher NAT yield in Indian donations, reported in an abstract[23] (1/121 yield versus ELISA and 1/179 yield versus chemiluminescent immunoassay); that site also performs their NAT in MP6 using the MPX2 test.

The current study demonstrates the high yield of NAT screening in India by minipool when using a test that is highly sensitive for HBV. MPX2 has the additional benefit of identifying the specific target responsible for the signal in the reactive samples, so no further discriminatory testing is required for reactive samples.[22],[24] Using the MPX2 test, a highly sensitive test with real time viral discrimination, in a minipool format provides both a high sensitivity for detection of HBV and more straightforward confirmation and target identification. Screening in minipool format provides the additional benefits of fewer tests performed, higher specificity and lower cost. The higher NAT yield in replacement donations compared to voluntary donations in this study is consistent with the previous observation by Hans et al. This highlights both the importance of NAT for detection of infections in donations and the value of building a volunteer donor base to maximize transfusion safety.[24]

Limitations of the study

Although some limitations in this study have been identified, mainly associated with the additional investigative work and follow-up studies that could be performed on the NAT yield donors, these do not detract from the key message. The NAT screening data generated are of high importance in determining the overall levels of NAT yield donations that could be identified, together with the important element of quantitation of these NAT yields.

  Conclusion Top

This is a large study, using pooled samples screened with a highly sensitive automated molecular screening system. The data generated clearly demonstrate the utility of the cobas TaqScreen MPX Test, version 2.0 performed in minipools and the importance of identifying potentially infectious donations before they enter the blood supply. This is of particular importance in such a large country, and where a significant proportion of the blood collected is processed into components: one donation may be used to provide a number of components that potentially could be transfused to multiple recipients.

The consistent finding of a high number of HBV yields in this study and most of the published Indian donor NAT screening studies suggests that HBV is the biggest infectious threat to the safety of the blood supply and measures that could be implemented to reduce this threat should be considered. To understand the phases of HBV infection being detected, additional testing and follow-up of NAT yield donors would be most useful.


The authors are obliged to team IMA Bareilly for providing necessary support. We acknowledge Dr. Ajit Sawhney, Ex-Chairman IMA Blood Bank for persuading to publish this data. We are thankful to Lab Technician Fauzia Q. Ansari in helping with data extraction and Jitendra Patel for compiling and processing of data. Last but not the least we appreciate the support of medical writer agency.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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