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 Table of Contents  
Year : 2019  |  Volume : 4  |  Issue : 2  |  Page : 191-197

Comparison of conventional immunochromatographic assay with new automated-Treponema pallidum hemagglutination assay for screening of syphilis in blood donors

1 Department of Transfusion Medicine, IMS and Sum Hospital, Bhubaneswar, Odisha, India
2 Department of Transfusion Medicine, Medanta-The Medicity, Gurgaon, Haryana, India
3 Department of Transfusion Medicine, Manipal Hospitals, Jaipur, Rajasthan, India

Date of Submission10-Sep-2019
Date of Acceptance21-Sep-2019
Date of Web Publication17-Oct-2019

Correspondence Address:
Dr. Aseem K Tiwari
Department of Transfusion Medicine, Medanta-The Medicity, Gurgaon, Haryana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/GJTM.GJTM_55_19

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Introduction: Serological tests for syphilis contributed greatly to the detection of Treponema pallidum infection in blood donors and especially in those who were missed during the medical selection. Although several different treponemal assays have been evaluated in India, the debate that which treponemal test-formats would be suitable for a particular blood transfusion service is far from settled. Aims and Objectives: The study was undertaken with an objective of comparing conventional immunochromatographic assay (ICA) with new automated T. pallidum hemagglutination assay (a-TPHA) in over 10,000 consecutive blood donor samples with fluorescent treponemal antibody-absorption assay (FTA-Abs) as the gold standard to resolve the discordant samples. Materials and Methods: The study was conducted in the Department of Transfusion Medicine in a large tertiary care hospital in India. Consecutive blood donors from July 2014 to January 2015 were evaluated simultaneously for antitreponemal antibodies by solid-phase ICA (SD BIOLINE Syphilis 3.0, Alere Medical Pvt. Ltd., USA), a-TPHA (Immucor Diagnostics, USA) and FTA-Abs (Biocientifica SA, FTA-Abs, Argentina). Performances of both the assays were evaluated using statistical tools. Results: A total of 10,129 donor samples were evaluated. A total of 113 (1.1%) donor samples were reactive for syphilis; either on ICA or a-TPHA or both. Ninety-one of these 113 reactive samples were reactive on FTA-Abs, giving seroprevalance of syphilis as 0.9%. a-TPHA was superior to ICA in terms of sensitivity, specificity, positive predictive value, negative predictive value, and Youden's index. Conclusion: The new “a-TPHA” is a superior assay to ICA as the screening assay for syphilis in blood donors.

Keywords: Automated Treponema pallidum hemagglutination assay, fluorescent treponemal antibody-absorption assay, immunochromatographic assay, rapid plasma reagin, serological test for syphilis, syphilis

How to cite this article:
Acharya DP, Tiwari AK, Aggarwal G, Arora D, Dara RC, Bhardwaj G, Gupta GK. Comparison of conventional immunochromatographic assay with new automated-Treponema pallidum hemagglutination assay for screening of syphilis in blood donors. Glob J Transfus Med 2019;4:191-7

How to cite this URL:
Acharya DP, Tiwari AK, Aggarwal G, Arora D, Dara RC, Bhardwaj G, Gupta GK. Comparison of conventional immunochromatographic assay with new automated-Treponema pallidum hemagglutination assay for screening of syphilis in blood donors. Glob J Transfus Med [serial online] 2019 [cited 2021 Dec 2];4:191-7. Available from: https://www.gjtmonline.com/text.asp?2019/4/2/191/269398

  Introduction Top

An effective blood transfusion service (BTS) has the responsibility to provide adequate and safe blood for better patient management. Safety is attained by recruiting low-risk donors, effective predonation screening, testing for transfusion-transmitted infections (TTIs), and judicious use of blood components. All the member states of the World Health Organization (WHO) are committed to provide access to safe blood to all and are bound by the World Health Assembly Resolutions, WHA28.72 in 1975 and WHA58.13 in 2005 with regard to safe blood access.[1],[2] Syphilis was one of the first TTIs to be tested in BTSs, and it still continues to be an important public health problem in some parts of the world. The WHO estimated that approximately 12 million new cases of syphilis are reported every year in the world, with more than 90% from developing countries.[3],[4] Fordyce reported the first case of transfusion-transmitted syphilis in 1915.[5] More than 100 cases have subsequently been reported in different countries including the USA and Great Britain.[6] However, no case of transfusion-transmitted syphilis has been reported from India.

In India, most centers use rapid plasma reagin (RPR) for screening of syphilis in blood donors because of its low cost, easy availability, and rules.[7],[8] However, increasingly, a larger number of centers are now adopting immunochromatographic assay (ICA) because of more objective reading of results and ease of use.[7] Both tests offer the advantage of quick testing with little equipment or expertise. However, both tests, RPR and ICA, have limitations of being resource-intensive and manual. Few centers have also begun using T. pallidum hemagglutination assay (TPHA) and enzyme-linked immune-assay (EIA).[9]

Aims and objectives

The lack of uniformity in screening method for syphilis among blood centers and lack of published comparative reports on different serological tests for syphilis (STS) from India encouraged the authors to study and compare the existing and widely used ICA with new automated TPHA (a-TPHA) as a screening test for blood donors and their comparison with the fluorescent treponemal antibody-absorption assay (FTA-Abs) considered as gold standard for the study.

  Materials And Methods Top


This was a prospective study conducted on blood samples from consecutive blood donors over 7 months from July 2014 to January 2015 in the Department of Transfusion Medicine at a large tertiary care hospital in Northern India. All blood donors who met the criteria for whole blood donation as laid down in the Drugs and Cosmetic Act[10] and provided informed written consent for blood donation were included in the study. Any donor who failed to fulfill the criteria was deferred with appropriate counseling and excluded from the study. Institutional guidelines and department standard operating procedures were followed for all processes and procedures.

Sample size calculation

In the present study, the prevalence of syphilis in healthy blood donors was assumed as 0.4%. Parameter of assessment was sensitivity (P) which was assumed as 90% for both the test methods with confidence level as 95% and precision (d) as + 10%.

Following formula was used for estimation of sample size (n = [Zα2 PQ]/d2).

where Zα = Value of standard normal variate corresponding to α level of significance, P = Likely value of parameter, Q = 1 – P, and d = Margin of errors which is a measure of precision.

The required sample size works out as 40 (reactive), and for obtaining 40 reactive samples, the number of donors to be screened was calculated as 10,000 (considering prevalence of 0.4%).

Donor classification

Donors were classified on the basis of gender, nature of donation, and frequency of donation. Nature of donation was either voluntary (donor who is not acquainted or related to any patient and has donated the blood on his/her own free will) or replacement (donor who is family member or friend). Frequency of donation classified donors as regular (donor who has donated at least twice in his/her lifetime and donated at least once in preceding 12 months) or occasional ( first time donor; or donor who has donated once in his/her lifetime before; or more than once but not in preceding 12 months).

Mandatory screening for transfusion-transmitted infection

All donors underwent mandatory testing for TTI (human immunodeficiency virus [HIV], hepatitis B virus, hepatitis C virus [HCV], syphilis, and malaria). Anti-HIV, hepatitis B virus surface antigen (HBsAg), and anti-HCV were performed on VITROS 3600 (Ortho Clinical Diagnostics, J and J, USA) using enhanced chemiluminescence technique. Test for Malaria (Microgene Diagnostics, India) and syphilis (Alere Diagnostics, USA) were done by solid-phase ICA.

Blood samples

As per the institutional policy, blood samples are collected in three pilot tubes during whole blood collection from the in-line sample pouch. 4 ml ethylene diamine tetra-acetic acid (EDTA; lavender-top), 6 ml EDTA (lavender top), and 4 ml clot accelerator (red-top) vacutainer were used for blood group serology, nucleic acid amplification test, and TTI, respectively.

Study algorithm of syphilis testing and reporting of results

In the present study [Figure 1], in addition to ICA, all consecutive blood donor samples were also tested for anti-treponemal antibodies by a-TPHA (Immucor Diagnostics, USA). Red-top vacutainer was used for performing ICA on the day of donation, while 6 ml lavender-top vacutainer was used for a-TPHA. All samples that tested initially reactive were re-tested by the same method before labeling the sample as “repeat reactive.”
Figure 1: Study algorithm

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All “repeat reactive” samples (either by ICA or a-TPHA) were confirmed by FTA-Abs, and discordance, if any, between these two tests (ICA and a-TPHA) would be resolved. FTA-Abs were considered as the gold standard for the study. Sample from all repeat reactive was aliqouted, labeled, and stored at −20 C for future FTA-Abs “batch” assay.

Automated treponemal hemagglutination assay

T. pallidum hemagglutination assay (TPHA, Immucor Diagnostics, US) is an indirect hemagglutination test for the detection of specific antibodies against T. pallidum. Avian erythrocytes are sensitized with antigen of the Nichol's strain of T. pallidum. In the presence of syphilitic antibodies (IgG and IgM), these test cells aggregate to form characteristic patterns on the surface of the microplate wells. This test was performed on the automated platform (Neo, Immucor Diagnostics, US).

Immunochromatographic assay

ICA (SD BIOLINE Syphilis 3.0, Alere Medical Pvt. Ltd, USA) was a membrane-based solid-phase assay for the qualitative detection of antibodies of isotypes; IgM and IgG against T. pallidum in serum, plasma, or whole blood. In the test procedure, recombinant T. pallidum antigen is coated on the test band region. Patient sample, when placed on the specimen pad, reacts with the antigen-colloidal gold conjugate. Running buffer facilitates the movement of the sample and antigen-colloidal gold conjugate along the length of the membrane to test region. If antibody to T. pallidum is present in the sample, an antigen-antibody-antigen complex will form. This is shown by a red-colored line in the test area. To serve as a procedural control, a red-colored line will always appear at the control region if the test has been performed correctly.

Florescent treponemal antibody-absorption test

FTA-Abs (Biocientifica Inmunofluor FTA-ABS, Argentina): T. pallidum subspecies pallidum was fixed on a microscope slide. Serum from reactive and nonreactive samples was diluted 1:5 in sorbent (to reduce nonspecific cross-reactivity) and was layered on that slide. If the patient's serum contains antibody, the antibody would coat the treponeme. Next, fluorescein isothiocyanate-labeled antihuman Ig was added; this combined with the patient's IgG and IgM antibodies that were adhering to treponeme and resulted in a visible test reaction when examined under fluorescence microscopy. This test was performed by the authors under the guidance of an expert of the National Institute of Pathology, New Delhi, who supervised the conduct and interpretation of FTA-Abs.

Statistical analysis

All the data were stored in Microsoft Excel Sheets (Microsoft Corporation, USA). The analysis included profiling of healthy donors on different demographic parameters such as age, gender, nature, and frequency of donation. ICA and a-TPHA test were evaluated and compared with respect to gold standard FTA-Abs assay on sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden's index using standard formulae. 2 × 2 cross-tables were generated manually and Chi-square test was used for testing of diagnostic test evaluation (SPSS software, Version 22.0, IBM, Bengaluru, Karnataka, India). For appropriate statistical evaluation, additional 100 known negative samples sourced from the study population (concordant negative; i.e., negative on both a-TPHA and ICA) were also tested with FTA-Abs along with all repeat reactive samples either on ICA, or a-TPHA, or both.

Ethical clearance

The “Medanta Institutional Ethics Committee” an independent ethics committee approved the study.

  Results Top

Demographics details of donors

A total of 10,129 donor samples were evaluated during the study period. There was male dominance with male-to-female being 14:1. The mean age of the study population was 36.9 years (range = 18–63 years; standard deviation = 8.83). Replacement donors constituted around 90.3% of total donor population. Almost 42.6% of total donors were regular donors [Table 1]. A total of 113 (1.1%) donor samples were initially as well as repeat reactive for syphilis; either on ICA or a-TPHA or both. None of the repeat reactive samples was reactive for other TTIs such as HBV, HCV, HIV, or malaria. Seroprevalance of syphilis in the present study was determined as 0.9% (91 confirmed reactive out of 10,129 donors). The prevalence of syphilis was lower in the regular blood donors as compared to occasional donors but was not statistically significant (not shown in tables).
Table 1: Demographics details of donors

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Comparison between immunochromatographic assay and automated Treponema pallidum hemagglutination assay with the gold standard (fluorescent treponemal antibody-absorption assay)

Of total 113 reactive samples, 51 samples were concordant reactive (i.e., reactive on both ICA and a-TPHA) whereas 62 samples were discordant (reactive on either ICA [n = 22] or a-TPHA [n = 40]). Therefore, the total number of repeat reactive samples with ICA and a-TPHA was 73 and 91, respectively. All the additional 100 known negative samples, which were included for statistically accurate comparison of ICA and a-TPHA with FTA-Abs, were nonreactive with FTA-Abs [Table 2].
Table 2: Spectrum of samples (concordant reactives, discordant reactives, and known negatives) assayed for fluorescent treponemal antibody-absorption assay

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The analytical sensitivity of ICA was 62% and the specificity was 90%. The analytical sensitivity and specificity of a-TPHA were higher than ICA; 87% and 94%, respectively. PPV – 92% versus 83% – and NPV – 90% versus 76% – of a-TPHA were significant higher than ICA. Youden's index of a-TPHA – 81 versus 52 – was also higher for a-TPHA as compared to ICA [Table 3]a, [Tables 3]b and [Tables 4].

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Table 4: Comparison between immunochromatographic assay and automated Treponema pallidum hemagglutination assay

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  Discussion Top

There has been much debate whether to retain or cease blood donor testing for syphilis. The Association for Advancing Transfusion and Cellular Therapies (formerly AABB) dropped the requirement in 1985, but the US-Food and Drug Administration did not support the proposal because syphilis testing was a potential surrogate for high-risk behavior for HIV infection. Even in 2000, it was felt that there were insufficient scientific data to warrant discontinuation of syphilis donor testing in the USA. Moreover, Brant et al.[11] reported that there has been a resurgence in syphilis infection in the United Kingdom general population (initially among men who have sex with men [MSMs]), and this has been reflected by an increased number of blood donors with evidence of early primary syphilis infection (as demonstrated by the presence of IgM antibodies). They also concluded that continued vigilance is required by blood services as the risk of syphilis increases in the general population. In their detailed factsheet on syphilis, the Center for Disease Control and Prevention, USA, also states that the rate of primary and secondary syphilis has been increasing among MSMs as well as heterosexual men and women.[12] In India, STS is mandatory. Most of the experts in India also recommend continuation of STS because it is a “lifestyle” marker of high-risk behavior.

In the present study, we evaluated the effectiveness of a new a-TPHA assay in comparison to our existing ICA assay as screening STS in blood donors. The demographics of the present study including male-to-female ratio and voluntary-to-replacement donor ratio among blood donors are similar to another Indian study performed in blood donors by Makroo et al.[13] The prevalence of syphilis, in the present study, with treponemal test as screening STS, is 0.9% and is similar to the results reported by Singh et al.,[14] who also used a treponemal assay. Other Indian studies, using nontreponemal assay as the screening STS, reported a prevalence of syphilis as 0.23%–1.3%.[13],[15],[16],[17]

The other debate so as to which STS is better, nontreponemal or treponemal, has tilted in favor of treponemal test.[18] This fact is also corroborated by Blood Bank External Quality Assessment Scheme (BEQAS, Jaipur) for blood banks in India. Over 500 blood banks in the country participate in this program, and almost 35% program participants use treponemal tests for blood donor screening (personal communication: Dr. Gajendra Gupta, Head BEQAS, Jaipur). However, these EQAS data are only a trend and may not be representative of entire BTS of the country. Several different treponemal assays have been evaluated in India,[9],[19],[20] the debate that which treponemal test-formats, ICA, EIA, chemiluminescence immunoassay (CLIA), manual-TPHA, or automated-TPHA, would be suitable for a particular BTS or for that matter the entire country is still less than settled.

Superiority of automated Treponema pallidum hemagglutination assay over immunochromatographic assay

The present study was done with a hypothesis that the new testing method (a-TPHA) is superior to the existing one (ICA). This study evaluated a-TPHA and ICA with FTA-Abs as the gold standard and the results proved the hypothesis; a-TPHA was superior to ICA with respect to sensitivity, specificity, PPV, NPV, and Youden's index. Higher sensitivity indicates that a-TPHA is better suited as a screening test than the ICA. Higher specificity offers an additional advantage of reducing unnecessary wastage of blood resource. Youden's index, a key indicator that combines both sensitivity and specificity of an assay also, suggests that a-TPHA is a better than ICA. The strength of association of a-TPHA with the gold standard is better in comparison to ICA.[21] A study by Wong et al.[22] that studied the efficiency of TPHA as screening test and found that TPHA could pick syphilis in every stage of syphilis even if they were negative by other screening assay and thus helpful in diagnosis and management.

Other advantages of automated Treponema pallidum hemagglutination assay

a-TPHA offers several other advantages such as automation, high throughput, shorter turn-around-time (TAT), and electronic transfer of results to hospital information system (HIS).

Automation with automated Treponema pallidum hemagglutination assay

The use of automation is a major consideration for BTS that performs a large number of screening tests (over 20,000 donations per annum). a-TPHA can be done in conjunction with blood grouping on the same equipment (Neo, Immucor Diagnostics, USA) and using the same EDTA donor sample. Bajpai et al.[23] mentioned that automation improves the objectivity and reproducibility of tests. It reduces transcription errors and human errors in donor identification. Documentation and traceability of tests, reagents, and processes and archiving of results are aother major advantages of automation. The WHO[24] also recommends the use of automation for screening of TTI in donated blood. From administrative perspective, saved workforce can be utilized for other blood bank jobs such as advanced immunohematological work-ups and clinical aspects of BTS such as active surveillance for adverse transfusion reactions.

Automating STS leads to better “line-balancing” in terms of process flow management. A constraint slows the process down and results in waiting for downstream operations. At present, STS and test for malaria by ICA represent a “constraint.” In blood banking if result of one assay (e.g., manual ICA) is delayed, it affects the TAT of completing the tests on donor sample and moving the blood units from “quarantined” inventory to “good-to-use” inventory. If this (a-TPHA) were to become routine, at least one of the constraints would go away resulting in better line-balancing.

High throughput with automated Treponema pallidum hemagglutination assay

Neo instrument can process 94 samples for a-TPHA at once and takes only around 90–100 min to interpret and process the results. As equipment, Neo also allows loading of additional samples (random access) without any interruption for the samples loaded earlier. This increases the productivity of the blood bank.

Shorter turnaround time with automated Treponema pallidum hemagglutination assay

The introduction of automated STS has improved the TAT. Previously measured TAT for ICA with a sample size of 80 was nearly 300 min starting from centrifugation of samples to the interpretation of results. It has fallen down significantly to 120 min for the same number of samples with a-TPHA. 180 min of person-hour could be saved and diverted for other work purpose.

Electronic interphase with automated Treponema pallidum hemagglutination assay

The results that are displayed on monitor screen of the machine can be transferred automatically through the HIS as donors' result. This adds significantly to the patient's safety as manual interference is reduced. Chances of error are reduced as every step is performed through prior tested software.

Limitations of automated Treponema pallidum hemagglutination assay

There are few limitations of a-TPHA; it is possibly suited for large blood banks (annual collection of 20,000 whole blood donations) with access to automated equipment.

Strengths of the study

The strengths of the study were the large sample size, use of FTA-Abs as the gold standard, and considering the repeat reactive STS samples for the analysis.


Subjective interpretation of FTA-Abs results was a limitation of the study.

Future directions

Besides a-TPHA, treponemal test is available on EIA and CLIA platforms. These assays (EIA/CLIA) offer all the advantage of a-TPHA such as shorter TAT and easier record keeping with an additional advantage that same pilot sample can be used for testing anti-HIV, anti-HCV, HbsAg, and STS on the same platform.

  Conclusion Top

The new “a-TPHA” is a superior assay than the ICA as screening method for syphilis in blood donors.


The authors would like to acknowledge technical support provided by Dr. Manju Bala of National Institute of Pathology, New Delhi, who guided the authors in performance and interpretation of FTA-Abs. We would also like to thank Immucor India for providing a-TPHA kits and Alere Medical Pvt. Ltd, USA, for providing FTA-Abs kit.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1]

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


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