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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 1  |  Page : 72-75

Gray zone sample testing in enhancing blood safety


Department of Transfusion Medicine and Immunohematology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India

Date of Submission02-Jan-2021
Date of Decision10-May-2021
Date of Acceptance10-May-2021
Date of Web Publication29-May-2021

Correspondence Address:
Dr. Murali Krishna Bogi
Department of Transfusion Medicine and Immunohematology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/gjtm.gjtm_2_21

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  Abstract 


Background: Gray zone samples with optical density (OD) lying between cutoff OD and 10% below the cutoff OD (cutoff OD × 0.9) were identified during routine Transfusion Transmissible Infections (TTI) screening. Enzyme-linked immunosorbent assay (ELISA) used for this purpose can sometimes fail to detect blood donors who are recently infected or possessing the low viremia. Estimation of a gray zone in ELISA testing and repeat testing of gray zone samples can further help in reducing the risks of TTI in countries where nucleic acid amplification testing for TTIs is not feasible. Methods: Repeat ELISA testing was done on gray zone samples in duplicate, the samples showing both OD values below gray zone were marked nonreactive, and samples showing one or both OD values in the gray zone were marked indeterminate. The samples on repeat testing showing one or both OD above cutoff values were labeled reactive. Results: Out of the 196 gray zone samples, 33 samples turned out to be reactive (as shown in the table), 39 samples were nonreactive, and 124 samples were again in the gray zone and were labeled as indeterminate. Conclusion: Estimation of gray zone samples with repeat testing can further enhance the safety of blood transfusion in resource-poor developing nations where more sophisticated and sensitive methods such as nucleic acid amplification test (NAT) is not available in all the blood banks.

Keywords: Enzyme-linked immunosorbent assay, gray zone, NAT, optical density, reactive, transfusion-transmissible infections


How to cite this article:
Kandukuri M, Vujhini SK, Bonagiri S, Bogi MK. Gray zone sample testing in enhancing blood safety. Glob J Transfus Med 2021;6:72-5

How to cite this URL:
Kandukuri M, Vujhini SK, Bonagiri S, Bogi MK. Gray zone sample testing in enhancing blood safety. Glob J Transfus Med [serial online] 2021 [cited 2021 Jun 25];6:72-5. Available from: https://www.gjtmonline.com/text.asp?2021/6/1/72/317124




  Introduction Top


The first step in reducing the risk of transmission of blood-borne infectious diseases is to select voluntary nonremunerated donors from low-risk populations who give blood on a regular basis as these individuals are at a lower risk of transmitting transfusion-transmissible infections than are family/replacement donors or paid donors. However, even with the most careful selection, some donors may be seropositive for HIV, hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV), syphilis, malaria, or other infectious agents. Therefore, rigorous screening of all donated blood is required to ensure the safety of the blood supply. The World Health Organization (WHO) recommends that all blood donations be screened for evidence of infection prior to the release of the blood and its components for clinical use. Screening of all blood donations should be mandatory for HIV, hepatitis B and C, malaria, and syphilis. Serologic testing for transfusion-transmitted diseases had historically been the foundation of blood screening, while newer strategies like nucleic acid testing (NAT) have helped further shorten the “window period.”[1] Currently, no technology exists to completely detect all window period donations. No matter how sensitive NAT becomes, we will never be able to completely close the exposure-to-seroconversion window period. Therefore, regardless of testing modality chosen, a nonzero risk of disease transmission still exists in all its seriousness.[2] Under current practice in India, potential blood donors, after registration, filling of donor health questionnaire, and brief medical examination, are sent for predonation counseling where process of blood donation, postdonation care, and the outcomes of donation are explained. After blood donation, pilot samples are collected for screening for p24 antigen and anti-HIV-1/2, anti-HCV, HBsAg, Venereal Disease Research Laboratory, and tests for malaria. It is very prudent to assess the utility of gray zone calculation and its role in improvising the current screening methodologies. We present here our experience of testing gray zone samples and its role in enhancing the sensitivity of current enzyme-linked immunosorbent assay (ELISA) technology used for blood donor screening at our setup.


  Materials and Methods Top


This prospective study was performed on blood donors coming for blood donation in the Department of Transfusion Medicine and Immunohematology, Nizam's Institute of Medical Sciences, Panjagutta, Hyderabad, Telangana State, India. It is a retrospective and prospective study involving 86,434 samples during the period January 2012 to December 2016. Eligible voluntary and replacement donors were included in this study after following donor selection criteria as laid down by the Drug and Cosmetic Act after taking informed consent from individual donors.[3] Immunoassay in the form of ELISA (manual method with automated washing and reading of optical density [OD]) was done for screening all donor blood samples strictly following the manufacturer's guideline. HIV and HBsAg screening were performed by fourth-generation ELISA kits, and for HCV, malaria, and syphilis, third-generation kits were used. Validation of each test was performed according to manufacturer's instruction. Quality control of ELISA testing was done by preparing Levey–Jennings (LJ) chart by simultaneously running the in-house borderline-positive controls for 30 consecutive runs as mentioned in the National Aids Control Organization guidelines.[4] Daily control data were plotted (E-ratio) on LJ chart, and there was a protocol to repeat the test if there are any outliers for that particular day. Any reactive samples which occurred in serial in a particular row or column were spotted, and the same samples were retested once again the next day with another technician. All the samples with OD more than the cutoff were considered reactive and blood units were discarded and donors were notified as per departmental standard operating procedure (SOP). Gray zone was calculated as 10% below the cutoff OD. All the samples with OD between cutoff value and 0.9 × cutoff value were marked as gray zone sample and were quarantined. All the gray zone samples were retested in duplicate for their respective viral marker using the same ELISA kits the next day. On repeat testing, the gray zone samples showing both OD values below 0.9 × cutoff value were marked as nonreactive and the blood units were included in the inventory. If on repeat testing, the gray zone sample showed one or both OD values above the cutoff value, it was marked as reactive and blood units were discarded and donor notified. The gray zone sample showing one or both OD values again as gray zone on repeat testing was marked as indeterminate and blood unit was discarded, but the donor was documented as nonreactive and notified for repeat testing after 3 and 6 months – if reactive, considered reactive.


  Results Top


Out of 86,434 samples tested for TTI's during the study period the combined reactivity for all the TTI's was found to be 1.87% (n=1619). Among all the TTIs, HBsAg reactivity was high (59%) when compared to the remaining TTIs. The year-wise reactivity is tabulated [Table 1].
Table 1: Overall incidence of TTIs

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The year-wise positivity for all TTIs was found to be 21.4% in 2012, 20.1% in 2013, 23.2% in 2014, 17.1% in 2015, and 18.1% in 2016, and the overall reactivity is 1.87%.

Out of the 15,721 donor units in the year 2012, 327 samples were reactive, 59 samples were in the gray zone, and the remaining were nonreactive and issued for the needy patients. Similarly, in 2013, 18,011 donor units were collected, 326 samples were reactive and discarded, 48 samples were in the gray zone, and the remaining were nonreactive and went to inventory. In the year 2014, 18,181 donor units were collected and 376 samples were reactive, 42 samples were in gray zone, and the remaining were nonreactive and went into inventory. In the year 2015, 16,194 donor units were screened for TTIs, 277 samples were reactive, 24 samples were in the gray zone, and the remaining units were nonreactive and went into the inventory. In 2016, 18,327 blood units were screened for TTIs, 293 samples were reactive and discarded, 23 samples were in the gray zone, and the remaining were nonreactive and issued according to the patients' needs [Table 2].
Table 2: Year-wise collection, reactive, and gray zone samples

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These gray zone samples (196) were tabulated year wise according to their positivity [Table 3].
Table 3: Year-wise gray zone samples with their corresponding TTI markers

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One ninety six samples were in the grey zone and were tested again the next day and results are shown in [Table 4].
Table 4: Repeat TTI testing of the gray zone sample

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Out of the 196 gray zone samples, 33 samples turned out to be reactive (as shown in the table), 39 samples were nonreactive, and 124 samples were again in the gray zone and were labeled as indeterminate. All the reactive samples were discarded according to the SOP. If on repeat testing, the gray zone sample showed one or both OD values above the cutoff value, it was marked as reactive and blood units were discarded and donor notified. The gray zone sample showing one or both OD values again as gray zone on repeat testing was marked as indeterminate and blood unit was discarded, but the donor was documented as nonreactive and notified for repeat testing after 6 months.


  Discussion Top


Blood transfusion is generally the process of receiving blood or blood products into patient's bloodstream intravenously. Transfusions are used for various medical conditions to replace lost components of the blood. Earlier days, transfusions used were of whole blood, but modern medical practice commonly uses only components of the blood, such as red blood cells, white blood cells, plasma, cryoprecipitate, and platelets.

Donated blood is usually subjected to processing after it is collected, to make it suitable for use in specific patient populations. Collected blood is then separated into blood components by centrifugation: red blood cells, plasma, platelets, albumin, clotting factor concentrates, cryoprecipitate, fibrinogen concentrate, and immunoglobulins.

The WHO recommends testing of all donated blood for transfusion-transmissible infections like HIV, HBSAg, HCV and Syphilis .Screening of donations for other infections, such as malaria or Chagas disease may be needed based on local epidemiological evidence.[5] According to the recent statistics of the WHO, 25 countries are not able to screen all donated blood for one or more infectious diseases and the main reason for this is because testing kits are not always available. However, the prevalence of transfusion-transmitted infections is much higher in low-income countries compared to middle- and high-income countries.[6]

HBV, HCV, and HIV are the most important agents responsible for TTIs and thus their testing on blood donors is mandatory worldwide due to potential serious clinical complications associated with these agents.[7],[8] With advances in screening techniques in the form of NAT, the risk of TTIs has decreased considerably.[9] Still, TTIs remain a threat to blood safety due to several factors such as genetic variations of infectious agents, presence of immunologically silent carriage, laboratory errors, and variations in the window period of the infectious agent, as well as limitations in screening testing methodology.[7] In developing countries where NAT test is not routinely practiced for screening due to nonaffordability, immunological assays like ELISA serve as the main screening tool in blood bank setup.

Several methods have been devised to improve the sensitivity of ELISA such as the inclusion of borderline reactive control samples in each run to minimize batch to batch, as well as day-to-day variation in testing. These borderline reactive samples are also able to detect minor variation in the assay procedure.[4] Another method to enhance the sensitivity of ELISA as a screening assay is an estimation of the sample lying in a gray zone and its repeat testing. It has been very well illustrated by Pereira et al.[10] that ELISA-based screening test for TTI in blood banks does involve a certain amount of uncertainty, especially around the cutoff zone used for calculating the reactive samples. Hence, they have emphasized the measurement of this uncertainty around the cutoff zone in the form of gray zone sample testing. At present, there are no such existing guidelines for gray zone sample testing in any regulatory authority in India and most of the blood banks in India follow the strategy I of one-time ELISA testing as a screening procedure as per the WHO guidelines.[4]

Gray zone sample testing might not have gained much relevance due to the issues of false positivity on repeat testing, wherein a study conducted in Turkey has reported a 70% false positivity on testing gray zone samples.[11] On repeat testing of gray zone samples by a confirmatory test, it was observed that 2 % of the samples were true positive or reactive.[12] We found a total of 119 (0.56%) samples in gray zone area for all three viral markers as compared to 0.14%–0.29% found by other authors.[12],[13] In our study, we had 196 gray zone samples which were tested and the total percentage was 0.22% when compared to other studies.

One of the limitations of our study is that we could not perform the confirmatory assay for individual markers. Hence, we are not able to comment on the overall effectiveness of repeat gray zone sample testing in improving the transfusion safety. However, repeat reactivity in gray zone sample testing is an alarming indication for mandatory implementation of more sensitive testing technologies like NAT in developing countries. In our study, we have detected 33 (16.84%) reactive samples on repeat testing, 124 samples in the gray zone (63.26%), and nonreactive samples on repeat testing (19.90%). After repeat testing of gray zone, the prevalence of TTIs increased by 0.3% which indicates the importance of the gray zone sample testing.

Although the risk of TTI's today is lower than ever, achieving zero risk transfusion still remains a challenge due to various emerging pathogens. Continuous improvement in the form of diligent donor screening and implementation of sensitive screening assay and effective pathogen inactivation procedures can ensure the elimination or at least reduction of the risk of acquiring TTIs.[14]

Implementation of cost-effective measures to improve the sensitivity of screening assays can be practiced, especially in areas where TTIs are highly prevalent without much-sophisticated equipment. Higher discard rate of reactive blood units and minor increase in cost due to new testing methodologies can be justified by the impact it would have in reducing the mortality and morbidity of patients due to TTIs in an already resource burden nation.


  Conclusion Top


Use of advanced technology like NAT for TTI screening is not feasible for all blood centers in developing countries owing to cost constraints. Estimation of gray zone samples with repeat testing can further enhance the safety of blood transfusion in such settings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Hans R, Marwaha N. Nucleic acid testing-benefits and constraints. Asian J Transfus Sci 2014;8:2-3.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Kucirka LM, Sarathy H, Govindan P. Wolf JH, Ellison TA, Hart LJ, et al. Risk of window period HIV infection in high infectious risk donors: systematic review and meta-analysis. Am J Transplant. 2011;11:1176-87.  Back to cited text no. 2
    
3.
Malik V. Drug & Cosmetics Act of India 1940. 13th ed. Lucknow, India: Eastern Book Company; 2001.  Back to cited text no. 3
    
4.
Sujatha Rao K. Guidelines on HIV testing. National Aids control organization, Ministry of Health and Family Welfare, India. 2007.  Back to cited text no. 4
    
5.
Screening donated blood for transfusion-transmissible infections recommendations. World Health Organization. 2009. ISBN 978 92 4 154788-8.  Back to cited text no. 5
    
6.
“Blood safety and availability Fact sheet 279”. World Health Organization. Retrieved 21 January 2016.  Back to cited text no. 6
    
7.
Kaur P, Basu S. Transfusion-transmitted infections: Existing and emerging pathogens. J Postgrad Med 2005;51:146-51.   Back to cited text no. 7
[PUBMED]  [Full text]  
8.
Bihl F, Castelli D, Marincola F, Dodd RY, Brander C. Transfusion-transmitted infections. J Transl Med 2007;5:25.   Back to cited text no. 8
    
9.
Schmidt M, Seifried E. Improving blood donor screening by nucleic acid technology (NAT) ISBT Sci Ser. 2010;5:219-29.  Back to cited text no. 9
    
10.
Pereira P, Westgard JO, Encarnação P, Seghatchian J, de Sousa G. The role of uncertainty regarding the results of screening immunoassays in blood establishments. Transfus Apher Sci 2015;52:252-5.   Back to cited text no. 10
    
11.
Acar A, Kemahli S, Altunay H, Kosan E, Oncul O, Gorenek L, et al. HBV, HCV and HIV seroprevalence among blood donors in Istanbul, Turkey: How effective are the changes in the national blood transfusion policies? Braz J Infect Dis 2010;14:41-6.  Back to cited text no. 11
    
12.
Chen D, Kaplan LA. Performance of a new-generation chemiluminescent assay for hepatitis B surface antigen. Clin Chem 2006;52:1592-8.   Back to cited text no. 12
    
13.
Bihl F, Castelli D, Marincola F, Dodd RY, Brander C. Transfusion-transmitted infections. J Transl Med 2007;5:25.  Back to cited text no. 13
    
14.
Verma A, Girish S, Chaudhary R. Grey Zone Samples Assessment in ELISA Screening of Blood Donors Enhances Blood Safety. 4th IAS Conference on HIV Pathogenesis, Treatment and Prevention. 2007.  Back to cited text no. 14
    



 
 
    Tables

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



 

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