| Abstract|| |
Several blood banks use grey zone (GZ) phenomenon (defined as samples with optical density within 10% below the cut off in enzyme immuno-assay [EIA]/chemiluminescence immunoassay [CLIA]) to further augment blood safety. There is paucity of data regarding usefulness of GZ sample and its application in Transfusion Transmissible Infection (TTI) screening procedures in blood transfusion services. We looked at our GZ sample results and their confirmatory test results to verify if it adds to blood safety in our set-up? We performed a prospective analytical study on blood donors' samples over two years. All the donors' samples were screened for TTI using CLIA. Samples with signal/cut-off ratio between ≥0.90 and <1.00 were classified under GZ. They were re-tested in duplicate and submitted to confirmatory testing: Neutralization Test for HBsAg, Immunoblot for HCV, and Western blot for HIV. Among the 50,064 blood donors donating the blood during study period, 573 (1.14%) donors were reactive for HBsAg, HCV, and HIV. Forty-seven (0.1%) TTI samples were GZ, but none was “confirmed positive.” The utility of GZ testing seems to be limited. However, this may be continued for sake of “erring on the side of caution” and since this only results in negligible wastage (0.1%) of blood units.
Keywords: Chemiluminescence immunoassay, confirmatory tests, grey zone, testing strategy, transfusion transmissible infection
|How to cite this article:|
Bhardwaj G, Tiwari AK, Arora D, Aggarwal G, Pabbi S, Setya D. Utility of grey zone testing strategy in transfusion transmissible infection testing in blood bank is of limited value!. Indian J Pathol Microbiol 2020;63:255-7
|How to cite this URL:|
Bhardwaj G, Tiwari AK, Arora D, Aggarwal G, Pabbi S, Setya D. Utility of grey zone testing strategy in transfusion transmissible infection testing in blood bank is of limited value!. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 Nov 27];63:255-7. Available from: https://www.ijpmonline.org/text.asp?2020/63/2/255/282691
| Introduction|| |
Integrated strategy for the provision of safe blood and blood products includes recruitment and retention of blood donors who are at low risk of transmitting infection, stringent donor screening consisting of meticulous donor history and examination, quality-assured screening of all donated blood units for transfusion transmissible infections (TTI), rational use of blood to reduce unnecessary transfusions, and the use of alternatives to transfusion, wherever possible. Screening of blood donors for infectious markers such as human immunodeficiency virus (anti-HIV), hepatitis B virus (HBsAg), and hepatitis C virus (anti-HCV) is commonly done by immunoassay in the form of antigen/antibody detection methods, such as enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immuno-assay (CLIA). Samples with optical density (OD) above or equal to cut-off OD are defined as reactive and samples below cut-off OD as nonreactive. Several centers also use Grey Zone (GZ) phenomenon (defined as samples with OD within 10% below the cut off) in a bid to further enhance blood safety.
However, there is paucity of data regarding usefulness of GZ sample and its application in TTI screening procedures in blood transfusion services. Therefore, we analyzed our GZ sample results, their re-test results, and confirmatory test results to verify if it adds to blood safety in our setting and its role in improvising the current screening methodologies.
| Materials and Methods|| |
It was a prospective analytical study performed on blood donors after undergoing medical selection as per departmental standard operating procedures (SOP) over a period of two years from January 2017 to December 2018. An informed consent was obtained from the donors stating that their blood samples would be tested for TTI such as HIV, HBV, HCV, Syphilis, and Malaria and they would be notified if the test results are reported positive.
All the donor's samples were screened using chemiluminescence immunoassay (CLIA), by an automated instrument, VITROS ECi (Ortho Clinical Diagnostics, USA) using sample from clot activator tube (pilot samples), collected during whole blood donation.
Cut-offs were automatically calculated by the machine.
Signal to Cut-off calculation:
A result of ≥1.00 indicates a reactive sample and the possible
presence of HBsAg/anti-HIV 1 + 2/anti-HCV.
A result of <0.90 indicates a nonreactive sample, negative for HBsAg/anti-HIV 1 + 2/anti-HCV.
A result of ≥0.90 and <1.00 indicates a borderline or GZ sample for HBsAg/anti-HIV 1 + 2/anti-HCV.
All the GZ samples were re-tested in duplicate for their respective viral marker using CLIA immunoassay on VITROS ECi.
Based on the results obtained on testing in duplicate of GZ samples, six possible scenarios were seen. These scenarios were assigned numbers, such as S1 (scenario 1), S2 (scenario 2), S3 (scenario 3), S4 (scenario 4), S5 (scenario 5), and S6 (scenario 6). On repeat testing, the GZ sample showing one or both S/C ratio of ≥1.00 was marked as reactive and it was assigned as S1. If one of the repeat tests was reactive (S/C ratio ≥1.00) and another repeat test were either in GZ (S/C ratio ≥0.90 and <1.00) or nonreactive (S/C ratio <0.9), they were marked as reactive and were categorised as S2 and S3, respectively. The GZ sample showing one or both S/C ratio again of ≥0.90 and <1.00 on repeat testing was marked as borderline or GZ and were assigned as S4. Similarly, GZ sample having one of the repeat tests again in GZ and second repeat as nonreactive was again marked as borderline or GZ and categorized as S5. On repeat testing, the GZ sample showing both S/C ratio <0.9 were marked as nonreactive and was assigned as S6. The possible scenarios obtained on repeat testing are depicted in [Table 1].
All GZ samples were further tested using confirmatory tests. For HBV, neutralization (Ortho clinical Diagnostics, Johnson & Johnson, USA) result was considered final. In case of HCV, immunoblot assay [(Desiscan HCV Plus), Bio-Rad, Hercules, CF, USA] result was considered final. While for HIV, Western blot (J. Mitra & Co. Pvt. Ltd, India) was the confirmatory test.
| Results|| |
Among the 50,064 blood donors donating the blood during two years of study, 573 (1.14%) donors were reactive for HBsAg, HCV, and HIV in the TTI screening tests and 47 (0.1%) blood donors' samples were GZ positive. Of the total 573 TTI-positive blood donors, 336 (0.67%) were positive for HBsAg, 138 (0.27%) for HCV, and 99 (0.2%) for HIV respectively. Donors having their TTI results in the GZ were 47, of which HBsAg was 05 (0.01%), HCV was 29 (0.06%), and HIV was 13 (0.03%), respectively.
Based on the results obtained on testing in duplicate of GZ samples, different combinations of results were seen. None of the GZ samples on repeat testing were in S1, i.e., reactive in both the repeats. Most of the results (24 samples) were in S4, i.e., in GZ and nonreactive on repeat testing. Seven samples came out to be in S6, i.e., nonreactive on both the repeat tests. The possible scenarios, interpretation, and results obtained on repeat testing of GZ samples are shown in [Table 1].
None of the 47 GZ samples further testing using confirmatory tests (neutralization for HBsAg, Western blot for HIV, and immunoblot for HCV), came out to be positive. The results are shown in [Table 2].
| Discussion|| |
In India, literature is scarce regarding confirmation of GZ positive samples and their significance in TTI screening procedures in a blood bank set up. There are studies, on GZ but have not used confirmatory tests for confirming the initial GZ reactive samples. Therefore, it becomes prudent to assess the utility of GZ calculation and its role in the current screening strategies. In this study, 0.1% (47/50064) TTI samples were in GZ, and on further evaluation of the results using confirmatory tests (neutralization for HBV/Western blot for HIV/immunoblot for HCV), none of them were “confirmed positive.” This proves negligible or nil value of GZ in current testing strategy. However, this may be continued as an “extra-precaution” and considering that overall number (1 in 1,000; 0.1%) that get discarded is quite small. However, it clear that GZ can never be used for “donor notification” since the probability of these tests being “confirmed positive” is next to impossible. This study was compared with other Indian studies,, analyzing the impact of GZ on TTI as shown in [Table 3]. In a study conducted by Solanki et al., on blood donors, they detected 119 (N = 21,252) of TTI samples to be in GZ. Out of these, 70 (58.8%) samples showed repeat reactivity on re-testing and only six (5.04%) were again GZ positive. In another study conducted by Anitha et al., a total of 144 (N = 21,908) blood donors were in GZ. On repeat testing of these GZ samples, 35 (24.30%) samples were found to be reactive for TTIs and 18 (12.5%) were in GZ. In our study, out of initial 47 samples, majority (37) of our GZ samples were again showing results in GZ (78.7%) on re-testing in duplicate. Only three GZ (6.3%) were reactive, in either of the repeat test (performed in duplicate) and none of these were positive in confirmatory tests. Lower rate of reactivity (n = 3; 6.3%) in this study as compared with other studies (58.8%, 24.30%), could be due to the fact that our testing methodology CLIA is far more precise than ELISA used in other two, published reports. Moreover, none of these studies performed confirmatory test on the GZ samples and therefore have limited value. High repeat reactivity (24.3–58.8%) was assumed “positive,” which seems to be completely incorrect as shown by “confirmation” test in this study.
|Table 3: Comparison of this study with other Indian studies in evaluating grey zone (GZ) samples|
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GZ results may be of limited relevance due to reported false positivity on repeat testing. A study conducted in Turkey reported 70% false positivity on re-testing GZ results. Study by Acar et al. estimated that on applying the confirmatory test to GZ samples resulted in only 2% confirmed positivity. These results are concordant with our results of GZ samples where eventually none was confirmed as “positive.” False-positive GZ is not limited to and could be due to cross-reactivity with other antibodies against other viruses, recent vaccination, molecular mimicry, etc.
Since none of the GZ samples were confirmed positive, GZ can be considered as initial reactive (false positive). This is further confirmed by their pattern of reactivity; larger number of HCV (0.06%), and HIV (0.03%) were GZ as compared with HBsAg (0.01%), which were fewer. Lower prevalence of a disease is associated with higher “false positivity.” In Indian population, prevalence of HCV (0.16%) and HIV (0.3%) are lesser as compared with HBsAg (1.18%).
| Conclusion|| |
The utility of GZ testing seems to be limited. However, this may be continued for sake of “erring on the side of caution” and since this only results in negligible wastage (0.1%) of blood units.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Aseem K Tiwari
Department of Transfusion Medicine, Medanta-The Medicity, Sector-38, Gurugram - 122 001, Haryana
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]