MANUAL ON SELECTION AND USE OF

INTERFERON-GAMMA
RELEASE ASSAYS (IGRAS)
FOR TESTING FOR TB INFECTION
ACKNOWLEDGEMENTS

Development of this manual was led by Wayne van Gemert

and Lucy Mupfumi of the Stop TB Partnership and Kaiser Shen
of the United States Agency for International Development
(USAID). The manual was drafted by Thomas Shinnick
(Independent Consultant) and underwent technical review
by the following members of the core group of the Global
Laboratory Initiative (GLI): Khalide Azam (Southern Africa
Tuberculosis and Health Systems Support Project of the East,
Central and Southern Africa Health Community), Roger
Calderón Espinoza (Partners in Health), Christopher Gilpin
(International Organization for Migration) and Marguerite
Massinga Loembé (Africa Centres for Disease Control
and Prevention; African Society for Laboratory Medicine).
Technical review was also provided by Amera Khan and
Akjagul Garajagulova of the Stop TB Partnership, and Sevim
Ahmedov, Cleophas D'auvergne, Peter Kerndt and Inoussa
Zabsonre of USAID.

All reasonable precautions have been taken by the authors

to verify the information contained in this publication.
However, the published material is being distributed without
warranty of any kind, either expressed or implied.
The responsibility for the interpretation and use of the material
lies with the reader. In no event shall the authors be liable
for damages arising from its use.

Development of this document was made possible with

financial support from USAID. The views expressed herein
are those of the authors and do not necessarily reflect those
of USAID or the U.S. Government.
ABOUT THIS MANUAL

This manual provides an overview of the recommended use

of interferon-gamma release assays (IGRAs) as a class
of in-vitro tests for the detection of TB infection and describes
the performance, steps for use and implementation
considerations for specific IGRAs that are either approved
by the WHO Global TB Programme or the Global Fund’s Expert
Review Panel for Diagnostics. This manual will be periodically
updated as additional tests become approved. Please contact
any of the authoring organizations to suggest any contributions
to this manual.

This manual is intended to inform Ministry of Health officials,

programme managers, testing site managers, quality
assurance unit personnel, supervisory laboratory staff
and technicians at national, state/provincial and testing site
level, as well as technical partners and donors.

VERSION 1 — OCTOBER 2022

COVER PHOTOS: © KENYA NTP, CHS AND PANGANI HOSPITAL NAIROBI, STOP TB PARTNERSHIP, ADOBE STOCK, UNSPLASH
ABBREVIATIONS

AIDS acquired immunodeficiency syndrome

BCG bacille Calmette-Guérin (vaccine)

BCT blood collection tube

CFP-10 culture filtrate protein 10

ELISA enzyme-linked immunosorbent assay

ESAT-6 early secretory antigenic target-6

HIV human immunodeficiency virus

IFN-γ interferon-gamma

IGRA interferon-gamma release assay

LMICs low- and middle-income countries

LTBI latent tuberculosis infection

PLHIV people living with HIV

POC Point of care

PPD purified protein derivative

QFT-Plus QuantiFERON-TB Gold Plus

SOP standard operating procedure

STBP/GDF Stop TB Partnership’s Global Drug Facility

TPT tuberculosis preventive treatment

TST tuberculin skin test

TB tuberculosis

TBF Standard E TB-Feron

WHO World Health Organization

TABLE OF CONTENTS

06 BACKGROUND AND SCOPE

09 WHO RECOMMENDATIONS ON TB INFECTION TESTING
09 TARGET POPULATION TO TEST
10 SELECTION OF TB INFECTION TESTS
11 SUMMARY

12 1. QUANTIFERON®-TB GOLD PLUS (QFT-PLUS) IGRA

23 2. WANTAI TB-IGRA
31 3. STANDARDTM E TB-FERON IGRA
40 4. T-SPOT®.TB ASSAY

48 COMPARISON OF IGRAS FOR THE DETECTION OF TB INFECTION

49 ANNEXES
54 SUGGESTED READING

05
BACKGROUND AND SCOPE
About one-fourth of the world’s population is estimated account the probability of progression to active TB
to be infected with Mycobacterium tuberculosis disease in a specific risk group, the epidemiology
(M. tuberculosis), the causative agent of tuberculosis (TB). and burden of TB, the availability of resources,
The vast majority of infected individuals show no signs or and the likelihood of a broad public health impact.
symptoms of TB and are not infectious, although they have
Testing for TB infection is desirable whenever feasible
an increased risk of progressing to active TB disease
to identify persons at the highest risk for developing
and becoming infectious. On average, about 5–10% of
active TB. However, it is not required in people living
those infected will develop active TB disease over the
with HIV/AIDS or in household contacts aged < 5 years,
course of their lives, most of them within the first five
particularly in countries with high TB incidence, given that
years after infection. The risk is particularly high among
the benefits of treatment (even without testing) clearly
children under the age of 5 years and among people
outweigh the risks. For individuals or populations with
with compromised immunity (e.g. people living with HIV).
a higher risk of harm due to TPT or (relatively) lower risk
TB infection (also called latent TB infection or LTBI) of progression to TB disease, the WHO recommendation
is operationally defined as a state of persistent immune states that confirmation of TB infection increases
response to stimulation by M. tuberculosis antigens with the certainty that individuals targeted for TPT would
no evidence of clinically manifest active TB disease. benefit from TPT. In particular, TB infection tests are
The WHO End TB Strategy has prioritized TB preventive recommended for HIV-negative children aged ≥5 years
treatment (TPT) for persons with TB infection who are and adults who are contacts of persons with active TB
at high risk of developing active TB disease as a key disease as well as other high-risk adults identified
component under Pillar 1, and in September 2018 for TPT. However, the current recommendations leave
at the first-ever United Nations High-Level Meeting ambiguity as they state that unavailability of TB infection
on Tuberculosis, Member States endorsed a political testing should not be a barrier to treat people who were
declaration committing to provide 30 million individuals judged to be at higher risk.
with TPT by 2022 to protect them from the development
There is no gold standard test for TB infection.
of active TB disease.
The current WHO-recommended tests for TB
Implementation and scale-up of TPT services require infection detection, skin tests (tuberculin skin test (TST)
strengthening of each element in the cascade and antigen-based skin tests) and Interferon-Gamma
of diagnosis and care starting from identification Release Assays (IGRAs), are indirect and require
of the target population to ruling out active TB disease the person to mount an immune response in order to
to testing for TB infection to the provision of preventive work properly.2 The tests measure the immune
treatment1 (FIGURE 1). sensitization to mycobacterial protein antigens
that occurs following M. tuberculosis infection.
Identification of target populations to test is critical
Whilst the earliest version of the TST measured a delayed
because population-wide TB infection testing prior to TPT
hypersensitivity reaction to exposure to Mycobacterium
in low- to middle-income countries (LMICs) is not feasible
purified protein derivative (PPD), newer, more specific
or cost-effective. That is because population-wide testing
versions have been developed that use M. tuberculosis
for TB infection has a high cost and the public health
(M.tb)-specific antigens CFP-10 and ESAT6.
impact is not well documented; the pooled prevalence
These new TB antigen-based skin tests include the
of positive results to the WHO-recommended tests for TB
Cy-Tb skin test (Serum Institute of India, India), Diaskintest
infection among people eligible for testing was reported
(JSC Generium, Russian Federation), and C-TST
to be 61% in LMICs.2 In addition, the available tests are
(Anhui Zhifei, Longcom Biopharmaceutical Co. Ltd,
imperfect and there is some risk of serious adverse drug
China). These tests combine the low cost of the TST
reactions during TPT. On the other hand, the benefits
and the high specificity of the IGRA.
of TPT are more likely to outweigh the harms in infected
individuals belonging to population groups in whom IGRAs measure the amount of interferon-gamma (IFN-γ)
the risk for progression to active disease significantly released in vitro by T-lymphocytes when mixed with
exceeds that of the general population. Children under M. tuberculosis-specific antigens ESAT6 and CFP-10 or
the age of 5 and PLHIV are two of those groups in which the number of T-lymphocytes producing IFN-γ following
the probability of progression is high and TPT stimulation with purified M. tuberculosis-specific antigens
benefits are very clear. Accordingly, WHO guidelines1 ESAT6 and CFP-10. The features of skin tests and IGRAs
on the detection and treatment of TB infection take into are compared in TABLE 1.

1. WHO consolidated guidelines on tuberculosis. Module 3: diagnosis. Tests for TB infection. Geneva: World Health Organization, 2022. https://www.who.int/publications/i/item/9789240056084
2. Alsdurf H, Hill PC, Matteelli A, Getahun H, Menzies D. The cascade of care in diagnosis and treatment of latent tuberculosis infection: a systematic review and meta-analysis. Lancet Infect Dis. 2016;16(11):1269–78

06
 FIGURE 1

ALGORITHM FOR TB INFECTION TESTING AND TB PREVENTIVE TREATMENT IN INDIVIDUALS AT RISK

HIV positive Household contact Other risk groupC

Any symptomA
Symptomatic?B
of current cough or fever
or weight loss or night sweats

No
No Yes Yes

<5 years 5 years +

Investigate for active TB

Skin test
No active TB or IGRA

Preventive treatment Positive

contraindicated?D Negative
or unavailable

Abnomal

Yes No CXRF

Defer preventive Give preventive Normal

treatment treatmentE or unavailable

Follow up for active TB as necessary, even for patients who have completed preventive treatment

A If <10 years, any one of current cough or fever, or history of contact with TB or reported weight loss or confirmed weight loss >5% since last visit
or growth curve flattening or weight for age <-2 Z-scores. Asymptomatic infants <1 year with HIV are only treated for LTBI if they are household
contacts of TB. TST or IGRA may identify PLHIV who will benefit most from preventive treatment. Chest radiography may be used in PLHIV on ART,
before starting LTBI treatment.
B Any one of the following symptoms: cough or fever or night sweats or hemoptysis or weight loss or chest pain or shortness of breath or fatigue.
Children < 5 years, should also be free of anorexia, failure to thrive, not eating well, decreased activity or playfulness to be considered asymptomatic.
C Other risk groups include silicosis, dialysis, anti-TNF agent treatment, preparation for transplantation or as defined by national guidelines.
D Contraindications include acute or chronic hepatitis, peripheral neuropathy (if isoniazid used), and regular and heavy alcohol consumption.
Pregnancy or a previous history of TB is not a contraindication.
E Regimen chosen based on considerations of age, strain (drug-susceptible or otherwise), risk of toxicity, availability, and preferences.
F CXR may have been carried out earlier on as part of intensified case finding.

WWW SOURCE: REPRODUCED FROM WHO CONSOLIDATED GUIDELINES ON TUBERCULOSIS1

www.who.int/publications/i/item/9789240056084

1. WHO consolidated guidelines on tuberculosis. Module 3: diagnosis. Tests for TB infection. Geneva: World Health Organization, 2022. https://www.who.int/publications/i/item/9789240056084

07
 TABLE 1
3
CHARACTERISTIC FEATURES OF SKIN TEST AND IGRA

SKIN TEST IGRA

TEST REQUIREMENTS

Requires intradermal administration of antigen into the Require fresh blood samples drawn by a trained
volar surface of the forearm. phlebotomist or equivalent.
Test must be read between 48–72 hours after Blood specimens must be incubated and tested within
the intradermal injection. 8–30 hours after collection.
Trained staff are required to administer and read skin Frequently IGRAs are performed in reference
induration. laboratories, (except newer tests that can be
Can be administered in a community setting or health performed near-POC) which may require an efficient
center. sample transport mechanism.

POTENTIAL INACCURACY

False-positive TSTs can result from contact Inaccurate results may result from a delay in specimen
with nontuberculous mycobacteria or vaccination transportation or errors in processing of blood
with bacille Calmette-Guérin (BCG).4 specimen.
Potential for inaccuracies and bias in reading skin False-negative results may occur due to
induration. immuno-suppressive conditions, faded immune
False-negative results may occur due memory, technical-operational variability,
to immunodeficiency conditions. and in children below two years of age.

ADVANTAGES

Single visit required to collect sample for conducting

Antigen-based skin tests have similar specificity the test.
to IGRAs. Results possible within 24 hours.
Can be performed in the field. No booster effect.
Significantly fewer resource needs compared to IGRA. No false-positive results due to BCG vaccination.
No laboratory testing required. For ELISA-based IGRAs: Use of automated platforms can
More familiar to practitioners in resource-constrained increase throughput and ensure greater test accuracy.
settings. For some IGRAs: Potential for connectivity, digital
recording and transmission of results.

CHALLENGES

Higher test cost.

Need for training in intradermal injection, reading For ELISA/ELISPOT assays: need for sophisticated
and interpretation. laboratory equipment, skilled laboratory personnel.
Second visit (by individual or health care worker) For near-POC IGRAs: run on specific proprietary equipment.
required for test reading. Need for persons trained in phlebotomy.
Recurrent global shortages and stock-outs of quality Second visit may be required to convey the test result
assured PPD. to the patient and make clinical decisions.
Skin tests require a cold chain. Potential for delays in sample transportation
Repeat test (two-step testing) for individuals whose to laboratories that offer IGRA testing.
immunity may have waned. If the laboratory SOP requires batching of tests
to reduce costs there may be delays in reporting results.

3. Adapted from WHO operational handbook on tuberculosis. Module 3: Diagnosis. Tests for tuberculosis infection. Geneva: World Health Organization, 2022. https://www.who.int/publications/i/item/9789240058347
4. Use of alternative interferon-gamma release assays for the diagnosis of TB infection: WHO policy statement. Geneva: World Health Organization, 2022. https://apps.who.int/iris/bitstream/handle/10665/351191/9789240042346-eng.pdf

08
WHO RECOMMENDATIONS
ON TB INFECTION TESTING1
Either a skin test or interferon-gamma release assay People living with HIV who have a positive test for TB
(IGRA) can be used to test for TB infection. infection benefit more from preventive treatment than
There is no strong evidence that one test should be those who have a negative TB infection test; TB infection
preferred overthe other in terms of predicting testing can be used, where feasible, to identify such
progression from TB infection to TB disease. individuals.

Neither skin tests nor IGRAs should be used in persons TB infection testing by skin tests or IGRA is not a
having a low risk of TB infection and disease. requirement for initiating preventive treatment in people
living with HIV or child household contacts aged < 5 years.

TARGET POPULATION FOR

SKIN TEST OR IGRA TESTING
AND TPT
WHO recommends two broad groups of at-risk → People with increased likelihood of exposure
populations for systematic assessment of eligibility to TB disease
and provision of TPT: • Household contacts of people with bacteriologically
confirmed TB, usually subdivided into:
→ People with an elevated risk of progression - children below five years of age,
from infection to TB disease - children five years and above, adolescents,
• People living with HIV. and adults.
• Patients suffering from silicosis, patients starting • Persons who live or work in institutional or crowded
or preparing for anti-tumor necrosis factor (TNF) settings, such as prisoners, health workers, recent
treatment, patients receiving dialysis, and patients immigrants from countries with a high TB burden,
preparing for organ or haematologic transplantation. homeless people and people who use drugs.

1. WHO consolidated guidelines on tuberculosis. Module 3: diagnosis. Tests for TB infection. Geneva: World Health Organization, 2022. https://www.who.int/publications/i/item/9789240056084

09
RECOMMENDATIONS FOR TB INFECTION TESTING BY GROUP*

PEOPLE WITH AN ELEVATED RISK OF PROGRESSION FROM INFECTION TO TB DISEASE

TB infection testing desirable but not required

People living with HIV
for initiating TPT

Patients suffering from silicosis, starting anti-TNF treatment, Systematic TB infection testing and treatment
receiving dialysis or preparing for transplantation recommended

PEOPLE WITH INCREASED LIKELIHOOD OF EXPOSURE TO TB DISEASE

Children below five years of age who are household TB infection testing desirable, but not required
contacts of people with bacteriologically confirmed TB for initiating TPT

Children five years and above, adolescents and adults who

TB infection testing recommended, but not required
are household contacts of people with bacteriologically
for initiating TPT
confirmed TB

Persons in institutional or crowded settings, such as

prisoners or health workers, recent immigrants Systematic TB infection testing and treatment
from countries with a high TB burden, homeless people may be considered
and people who inject drugs.

*Note that WHO does not recommend the use of skin tests or IGRA for the diagnosis of active TB disease.

SELECTION OF TB INFECTION
TESTS
A positive test result by either method is not by itself implement testing for TB infection, taking into account
a reliable indicator that the person will progress to TB the benefit of preventive treatment, the risk of over
disease, and may be followed by further diagnostic or under treatment of contacts who are NOT tested
evaluation including chest X-ray or a mWRD test against the costs and logistical difficulties to identify
to exclude active TB as needed and available (FIGURE 1). persons who are infected and would benefit most from
Conversely, a negative skin test or IGRA test result does TPT. While neither skin tests nor IGRAs have proven useful
not rule out TB infection, given the possibility of a to predict, among those who test positive, who will
false-negative test result among at-risk groups, such as develop active TB, the risk of progression is believed
young children or among those recently infected. to 5-10% over a lifetime, and higher among individuals
National health authorities need to decide how to who are infected and immunocompromised.

10
SUMMARY
The initial WHO recommendations on the use of IGRAs on the pathway to potential WHO endorsement.
were based on evidence from evaluations of
This implementation manual describes the operational
the QuantiFERON®-TB Gold In-Tube (QIAGEN GmbH,
steps and implementation considerations for use of IGRAs
Hilden, Germany) and T-SPOT®.TB IGRAs (Oxford
approved by WHO or the Global Fund’s Expert Review
Immunotec Ltd, Abingdon, UK).5 In 2021, WHO reviewed
Panel for Diagnostics: as of the time of publication of this
the evidence on the performance of the next-generation
manual, this comprises the QFT-Plus IGRA, the QIAreach
of QIAGEN’s QuantiFERON-TB IGRA, QuantiFERON®-TB
QFT IGRA, the WANTAI TB-IGRA, the TB-Feron IGRA
Gold Plus (QFT-Plus), and the WANTAI TB-IGRA (Beijing
and the T-Spot.TB IGRA.
WANTAI Biological Pharmacy Enterprise Co., Ltd, Beijing,
China), and in January 2022 updated its recommendations In October 2022, QIAGEN paused the commercialization
for the use of IGRAs for the detection of TB infection to of QIAreach QFT, a near-POC IGRA. This manual will be
include the use of the QIAGEN QFT-Plus and the Beijing updated when this product becomes available again
WANTAI TB-IGRA.6 from QIAGEN.
Based on available data at the time of WHO review As of the time of publication of this manual, QFT-Plus
in 2021, the QIAreach® QuantiFERON®-TB (QIAreach QFT) reagents and T-Spot.TB reagents are available in the
IGRA, T-SPOT®.TB 8 with T-Cell Select (Oxford Stop TB Partnership GDF catalog.
Immunotec) and the StandardTM E TB-Feron (TBF)
IGRA (SD Biosensor, Gyeonggi-do, Republic of Korea) A detailed discussion about the processes for implementing
could not be adequately compared with the any new diagnostic test is available in Module 3
WHO-recommended IGRAs. QIAreach QFT and TBF of the WHO operational handbook on tuberculosis.7
have been reviewed and approved as Risk Category 2 A detailed discussion about implementing a TPT program
by the Global Fund’s Expert Review Panel for Diagnostics and TB infection testing is available in Module 1
(ERPD), which acts as an interim approval mechanism of the WHO operational handbook on tuberculosis.5

ERPD APPROVAL FOR TB DIAGNOSTICS: WHAT IS IT?

The Global Fund's Expert Review Panel for Diagnostics (ERPD) is a group of independent experts who review
the potential risks and benefits associated with the use of diagnostice products and make recommendations
to the Global Fund and Unitaid on their use. The WHO Regulation and Prequalification Department hosts the ERPD.

ERPD approval of TB diagnostics is intended as an interim approval mechanism on the pathway to potential WHO
endorsement, either as a WHO Global TB Programme recommendation or by WHO prequalification. ERPD approval
allows countries to use Global Fund funding to procure products for a time-limited period, with possibility for renewal.

The current list of TB diagnostics approved by ERPD can be found on the Global Fund eligible products lists. ERPD
approval of product is categorized as either Risk category 1 or 2. While products in boh categories meet established
ERPD standards around manufacturing site quality and risk management systems, and have adequate evidence
of analytical performance, Risk category 2 products have limited clinical performance data in the settings of intended
use and/or limited stability data to assign shelf-life.

5. WHO operational handbook on tuberculosis. Module 3: Diagnosis. Tests for TB infection. Geneva: World Health Organization, 2022. https://www.who.int/publications/i/item/9789240058347
6. Use of alternative interferon-gamma release assays for the diagnosis of TB infection: WHO policy statement. Geneva: World Health Organization, 2022. https://apps.who.int/iris/bitstream/handle/10665/351191/9789240042346-eng.pdf
7. WHO operational handbook on tuberculosis. Module 3: diagnosis. Rapid diagnostics for tuberculosis detection. Geneva: World Health Organization, 2021.
https://www.who.int/publications/i/item/who-operational-handbook-on-tuberculosis-module-3-diagnosis---rapid-diagnostics-for-tuberculosis-detection

11
 1.
QUANTIFERON-TB®
GOLD PLUS
(QFT-PLUS) IGRA

14 PRINCIPLES OF THE ASSAY

15 PERFORMANCE
15 EQUIPMENT TO PERFORM THE ASSAY
16 HOW TO PERFORM THE ASSAY
19 OPERATIONAL CONSIDERATIONS

12
 QUANTIFERON-TB® GOLD PLUS
(QFT-PLUS) IGRA

QUANTIFERON-TB® GOLD PLUS (QFT-PLUS) IGRA

The initial WHO 2018 recommendations for the populations in conjunction with risk
use of IGRAs for the detection of TB infection assessment, radiography, and other
were based, in part, on evidence from the medical and diagnostic evaluations.
QuantiFERON®-TB Gold In-Tube assay There are no known population restrictions
(QIAGEN GmbH, Hilden, Germany). However, for the use of the QuantiFERON IGRAs.
the manufacturer discontinued the production
The tests rely on two main phases (FIGURE 2).
of the QuantiFERON-TB Gold In-Tube assay
In the first phase, whole blood is collected
and replaced it with the QuantiFERON®-TB
into specialized blood collection tubes (BCTs)
Gold Plus (QFT-Plus) assay. In the 2022
containing antigens or controls, incubated
recommendations, WHO concluded that the
at 37°C to elicit IFN-γ production by CD4
current recommendations for the use of IGRAs
and CD8 lymphocytes and centrifuged
for the detection of TB infection are also valid
to harvest plasma. In the second phase,
for the QFT-Plus assay. However, there was
the amount of IFN-γ present in the harvested
insufficient evidence to compare the
plasma is measured to identify in vitro
QIAreach® QuantiFERON®-TB (QIAreach QFT)
8 responses to the peptide antigens that are
with the WHO-recommended IGRAs.
associated with M. tuberculosis infection.
Only the QFT-Plus is discussed in this manual as
QIAGEN has paused the commercialization of The QFT-Plus assay collects whole blood into
QIAreach QFT. The QFT-Plus assay is available 4 BCTs. Two tubes contain antigenic peptides
in the Stop TB Partnership GDF catalog. of ESAT-6 and CFP-10 (TB1 and TB2 tubes)
and two tubes contain controls (Nil tube,
The QuantiFERON-TB IGRAs are in vitro
Mitogen tube). An enzyme-linked
diagnostic tests that use peptides from
immunosorbent assay (ELISA) is used to detect
the ESAT-6 and CFP-10 proteins to stimulate
the amount of IFN-γ in the harvested plasma.
cells in heparinized whole blood followed
by the detection of IFN-γ to measure in vitro Details of the holding times and temperatures
responses to those peptide antigens. These are described later in this document.
IGRAs are indirect tests for M. tuberculosis The specimen transport must comply with the
infection and are intended for use in at-risk holding times and temperature requirements.
WWW https://www.theglobalfund.org/media/9461/psm_productsdiagnosticstb_list_en.pdf

SOURCE: REPRODUCED FROM QIAGEN KIT INSERT

FIGURE 2

WORKFLOW FOR THE QFT-PLUS

37°C

Centrifugation Plasma
Blood collection Imune stimulation Time to perform test
15min at 2000 Up to 28 days at
4 ml 37°C, 16-24hr 2 to 3 hours
to 3000 RCF (g) 2-8°C or -20°C

8. Use of alternative interferon-gamma release assays for the diagnosis of TB infection: WHO policy statement. Geneva: World Health Organization, 2022. https://apps.who.int/iris/bitstream/handle/10665/351191/9789240042346-eng.pdf

13
 QUANTIFERON-TB® GOLD PLUS
(QFT-PLUS) IGRA

PRINCIPLES OF THE ASSAY

Whole blood specimens are collected into each of to earlier versions of the test.
the four QFT-Plus blood collection tubes. The tubes differ
by which antigens or controls have been dried onto The contents of the QFT-Plus blood collection tubes must
the walls of the tube. Alternatively, blood may be be thoroughly mixed with the collected blood to ensure
collected in a single vacutainer tube that contains heparin that the antigens on the tube walls are completely
as the anticoagulant and then transferred to dissolved. The tubes are then incubated in an upright
the QFT-Plus blood collection tubes. position at 37°C for 16 to 24 hours, during which time
The Nil tube adjusts for background (e.g. excessive levels the immune stimulation occurs. The samples are
of circulating IFN-γ or presence of heterophile then centrifuged, the plasma is removed and the amount
antibodies). The IFN-γ level of the Nil tube of IFN-γ (IU/ml) is measured by ELISA.
is subtracted from the IFN-γ level for the TB Antigen tubes
RESULT INTERPRETATION
and Mitogen tube.
A QFT-Plus assay is considered positive if the IFN-γ
The Mitogen tube is a positive control for the ability
response to either TB antigen (TB1 or TB2) tube
of the lymphocytes to produce IFN-γ and also serves
is significantly above the Nil IFN-γ IU/ml value;
as a control for correct blood handling and incubation.
specifically, if the Nil value is ≤ 8.0 IU/ml and either TB
The TB1 tube contains long peptides from ESAT-6 antigen tube minus the Nil IFN-γ value is ≥ 0.35 IU/ml
and CFP-10 that are designed to stimulate IFN-γ and at least 25% of the Nil value, irrespective of
production from CD4+ T-helper lymphocytes. the mitogen minus Nil value. The plasma sample from
The TB2 tube contains the same CD4 antigens of TB1 the Mitogen tube serves as an IFN-γ positive control
and an additional proprietary set of short peptides for each specimen tested. The test is considered
designed to stimulate IFN-γ production from CD8+ indeterminate if the mitogen response is <0.5IU/ml,
cytotoxic T lymphocytes. The detection of IFN-γ together with both TB antigen responses <0.35IU/ml
production by CD8 T cells appears to increase or ≥0.35IU/ml and <25% of Nil value, or the Nil response
sensitivity for the detection of TB infection compared >8.0IU/ml. (TABLE 1)

INTERPRETATION CRITERIA FOR QUANTIFERON-TB GOLD PLUS (QFT-PLUS)

RESULTS NIL TB RESPONSE INTERPRETATION

TB1 and/or TB2 minus Nil ≥0.35 and ≥25% of M. tuberculosis infection
Positive ≤8.0
Nil is likely

Mitogen minus Nil <0.5, and TB1 and TB2 M. tuberculosis infection
Negative ≤8.0
minus Nil <0.35 or ≥0.35 and <25% of Nil is NOT likely

>8.0 Any
Likelihood of M. tuberculosis infection
Indeterminate
TB1 and TB2 <0.35 or ≥0.35 and <25% of Nil cannot be determined
≤8.0
and Mitogen minus Nil <0.5

SOURCE: REPRODUCED FROM IMAGE PROVIDED BY SYLVIA M. LACOURSE9

9. Sylvia M et al. (2022). Latent Tuberculosis Infection. National HIV curriculum. https://www.hiv.uw.edu/go/co-occurring-conditions/latent-tuberculosis/core-concept/all

14
PERFORMANCE
Because there is no gold standard test to diagnose TB performance of the test in those with microbiologically
infection, direct measurement of the sensitivity confirmed active disease.
and specificity of the skin tests and IGRAs to assess TB
infection is not possible, therefore, surrogate measures Using this approach, the manufacturer showed that
have been used. The test´s specificity is estimated the QFT-Plus test has a sensitivity of 94.1%
by evaluating its performance in people at low risk (398/423 individuals) for detecting persons known
of having TB infection. This frequently involves testing to have active TB disease and a specificity of 97.2%
healthy individuals in settings with a very low (713/733 individuals)10 for correctly identifying persons
prevalence of TB who have no known risk factors for TB thought not to have TB infection. The indeterminate rate
infection. Sensitivity is approximated by evaluating the was less than 3% in these populations.

EQUIPMENT TO PERFORM THE ASSAY

MATERIALS REQUIRED BUT NOT PROVIDED MATERIALS PROVIDED

Phlebotomy materials QuantiFERON Nil Tubes (gray cap with white ring)

Calibrated variable volume pipettes for delivery QuantiFERON TB1 Tubes (green cap with white ring)
of 10 μl to 1000 μl with disposable tips
QuantiFERON TB2 Tubes (yellow cap with white ring)
Calibrated multichannel pipette capable of delivering
50 μl and 100 μl with disposable tips QuantiFERON Mitogen Tubes (purple cap with white ring)

Microplate shaker capable of speeds between QFT-Plus ELISA kit

500 and 1000 rpm

Deionized or distilled water, 2 liters

Microplate washer (automated washer recommended)

Microplate reader fitted with 450 nm filter

and 620 nm to 650 nm reference filter

37°C ± 1°C incubator (CO2 not required)

Refrigerator 2°C-8°C

Timer NB: QFT-Plus can be automated on the Dynex DS2

platform or the DiaSorin LIAISON XL and XS
Centrifuge capable of centrifuging blood tubes
platforms to simplify testing and interpretation
at least to 3000RCF (g) for plasma separation
of IGRA results (FIGURE 3).

10. QFTPlus-IGRA package insert.

15
 QUANTIFERON-TB® GOLD PLUS
(QFT-PLUS) IGRA

HOW TO PERFORM THE ASSAY

(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

PHASE 1 : BLOOD COLLECTION, ANTIGEN STIMULATION AND HARVESTING OF PLASMA

1 2 3
INCUBATION AND HARVESTING
SPECIMEN COLLECTION MIXING OF TUBE CONTENTS OF PLASMA

Label each tube appropriately Immediately after filling the QFT-Plus Incubate QFT-Plus BCTs UPRIGHT
and record the date and time BCTs, gently mix by inverting them at 37°C ± 1°C for 16 to 24 hours.
of sample collection. ten (10) times to make sure the entire The incubator does not require CO2
inner surface of the tube is coated or humidification.
For each patient, collect 1 ml of blood with blood. This will dissolve antigens After incubation, the tubes may be
by venipuncture directly into each on tube walls. held between 4°C and 27°C for up
of the QFT-Plus BCTs. to 3 days prior to harvesting of the
This procedure should be performed Transfer the QFT-Plus BCTs to a 37°C plasma.
by a trained phlebotomist or ± 1°C incubator as soon as possible
equivalent health worker competent and no more than 16 hours after Harvesting of plasma is facilitated by
in blood collection procedures. sample collection. Prior to incubation centrifuging the tubes at 2000–3000
Alternatively, draw blood into at 37°C ± 1°, maintain the tubes x g (RCF) for 15 minutes.
a lithium/sodium-heparin tube at room temperature (22°C ± 5°C). It is possible to harvest the plasma
(minimum volume 5 ml) NB: blood collected in lithium without centrifugation, but additional
and dispense 1 ml into each or sodium heparin tubes must be care is required to remove the
of the 4 QFT-Plus BCTs. maintained at room temperature plasma without disturbing the cells.
for no more than 12 hours from the
Harvest plasma samples using
time of blood collection to transfer
a pipette. Avoid pipetting up and
to BCTs and subsequent incubation.
down or mixing the plasma by any
Should the heparin tubes need to be means prior to harvesting. At all
refrigerated after sample collection, times, take care not to disturb the
the following steps should be material on the surface of the gel.
followed in the sequence specified: Plasma samples can be stored in
The tubes must be kept at room centrifuged blood collection tubes
temperature between 15 minutes for up to 28 days at 2°C to 8°C.
and 3 hours after collection. Harvested plasma samples can also
be stored below –20°C for extended
The blood sample can then be
periods.
stored at 2-8°C for 16 to 48 hours.
Allow tubes to equilibrate at room
temperature prior to transfer
to BCTs.
Aliquoted BCTs should be placed
in the 37oC incubator within
2 hours of blood transfer.
NB: The total time from blood draw
into heparin tube to 37°C incubation
must not exceed 53 hours.

16
HOW TO PERFORM THE ASSAY (CONTINUED)
(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

PHASE 2 : DETECTION OF IFN-γ AND GENERATION OF RESULTS

4 5 6

PREPARE IFN-γ STANDARDS PERFORM QFT-PLUS ELISA CALCULATE RESULTS

Reconstitute the IFN-γ Standard with Add 50 μl of the working-strength Calculate results using the most
the volume of deionized or distilled conjugate to each well of the current version of the
water indicated on the label QFT-Plus ELISA plate. QuantiFERON-TB Gold Plus Analysis
of the vial. Software for your region.
Mix gently. Add 50 μl of test plasma
This generates a final concentration or standards to the appropriate
of 8.0 IU/ml. wells of the QFT-Plus ELISA plate

Label 4 tubes “S1”, “S2”, “S3”, and “S4”. Incubate for 120 ± 5 minutes at room
temperature (22°C ± 5°C).
Add 150 μl of Green Diluent to S1, S2,
S3, S4. Wash the ELISA plate wells with
400 μl wash buffer at least 6 times.
Add 150 μl of the kit standard to S1
and mix thoroughly. Add 100 μl of substrate solution.
Mix using a shaker set at a speed
Transfer 50 μl from S1 to S2 and mix not more than 900 rpm.
thoroughly.
Incubate for 30 minutes at room
Green Diluent alone serves temperature.
as the zero standard (S4).
Add 50 μl of stop solution.
Mix using shaker.

Read absorbance at 450 nm with

a 620 to 650 nm reference filter.

WWW QuantiFERON-TB Gold Plus Analysis Software for your region, available for download at www.quantiferon.com

17
 QUANTIFERON-TB® GOLD PLUS
(QFT-PLUS) IGRA

FIGURE 3

WORKFLOW OF THE QFT-PLUS IGRA

Following sample collection in QFT-Plus blood collection to the testing lab at 4-27°C within 3 days. Note that
tubes, the sample should be transported to the testing lab automation is possible on the DiaSorin LIAISON XL and
and incubated within 16 hours of collection. Alternatively, XS platforms using the LIAISON QFT-Plus detection assay.
stimulation can be performed at the sample collection Results can be viewed via the LIAISON Quantiferon
site (37°C for 16-24h), then samples are transported Software (LQS) or the laboratory information system (LIS).

Flexible blood
collection option

Li/Na Hep single-tube option Innovative CD8+

technology

37°C

Send to Lab Send to Lab Simple and fast T cell incubation

37°C

Dispensing into QFT-Plus Simple and fast T cell incubation Send to Lab
Blood Collection Tubes

Centrifugation

CLIA LIAISON QFT-Plus Plasma ELISA QFT-Plus

LQS LIS

LQS® LIS connection QuantiFERON SW

(optional) (optional)

WWW SOURCE: REPRODUCED FROM QIAGEN QUICK GUIDE

www.qiagen.com/dk/applications/tb-management/workflow

18
CASE STUDY: SELECTION OF AN AUTOMATED ELISA SYSTEM

The International Organization for Migration (IOM) performs IGRA testing for selected migrant and refugee
populations as part of health assessments performed on behalf of destination governments. IOM currently
operates 35 laboratories across Africa, Asia and the Middle East. Where the caseload for performing IGRA
testing is high, the Qiagen QuantiFERON Gold Plus assay is used. In order to standardise testing across
our laboratories, the Dynex DS2 automated ELISA system is used to perform the test.
Test accuracy depends on the generation of an accurate standard curve and the results derived from
the standards must be examined before test sample results can be interpreted. The Dynex DS2 is one
automated and programmable ELISA solution that allows for the automated preparation of a standard curve and
the calculation of the in vitro responses to the peptide antigens that are associated with M. tuberculosis infection from
individuals’ blood samples.
One limitation with use of an automated ELISA system is the need for regular periodic maintenance. Although
maintenance can be performed by QIAGEN and their local distributors, it is an additional cost that needs to be
considered and budgeted for, when choosing the Dynex DS2 solution.

WWW www.dynextechnologies.com/our-products/ds2

OPERATIONAL CONSIDERATIONS

CONSIDERATIONS Because of the need for sophisticated laboratory equipment and highly
FOR SITE SELECTION skilled laboratory personnel to perform the ELISA, the test is best suited
for implementation in intermediate or national reference laboratories.
Because of the use of ELISAs for other diseases, national programs may
be able to leverage collaboration with other, non-TB-specific laboratories,
having the capacity for blood draws and ELISA testing. The biosafety
precautions for the QFT-Plus test are the same as most tests that are
performed in BSL-2 laboratories.

If the test is performed at a centralized site, it will be necessary to establish

efficient specimen transport systems to ensure that blood samples can be
transported from peripheral sites to the IGRA testing laboratory within
the recommended time frames. For QFT-Plus, blood samples may be
transported at room temperature (22 ± 5°C) or transported under cold chain
(2-8°C) to the centralized site. The maximum time between blood collection
in QFT-Plus tubes and processing at the testing laboratory for room
temperature blood samples is 16 hours. For cold chain storage, blood must
be collected and placed under cold chain in a single lithium heparin tube,
and later aliquoted into the QFT-Plus BCTs. When under cold chain, this allows
the maximum time between collection and processing to be extended
to 53 hours.

TIME REQUIRED The time required to perform the QFT-Plus ELISA is estimated below;
FOR PERFORMING ASSAY the time of testing multiple samples when batched is also indicated:
→ 37°C incubation of blood tubes: 16 to 24 hours.
→ ELISA: Approx. 3-3.5 hours for one ELISA plate.
• A 96-well ELISA plate can contain samples from up to 22 individuals.
• <1 hour of labor.
• Add 10 to 15 minutes for each extra plate.

19
 QUANTIFERON-TB® GOLD PLUS
(QFT-PLUS) IGRA

STORAGE → Blood collection tubes

AND HANDLING • Store blood collection tubes at 2°C to 25°C.
• Minimum remaining shelf life at the time of order readiness as per GDF
agreement with QIAGEN: 10 months.

→ Kit reagents
• Store kit reagents at 2°C to 8°C.
• Always protect Enzyme Substrate Solution from direct sunlight.
• Maximum remaining shelf life as per GDF agreement with QIAGEN:
24 months.

→ Reconstituted and unused reagents

• The reconstituted kit standard may be kept for up to 3 months if stored
at 2°C to 8°C.
• Once reconstituted, unused Conjugate 100x Concentrate must be returned
to storage at 2°C to 8°C and must be used within 3 months.
• Working strength conjugate must be used within 6 hours of preparation.
• Working strength wash buffer may be stored at room temperature for up
to 2 weeks.

FORECASTING The Stop TB Partnership’s GDF has negotiated concessional pricing

AND ORDER PLANNING of US$15.90 per patient sample for the QFT-Plus IGRA, for the following
eligible countries:
→ All low- and middle-income countries except Turkey.
→ Listed high-income countries:
Antigua and Barbuda, Bahamas, Barbados, Chile, Estonia, Guam,
Hong Kong SAR China, Israel, Latvia, Lithuania, Macao SAR China,
New Caledonia, Palau, Panama, Seychelles, St. Kitts and Nevis,
Taiwan China, Trinidad and Tobago, Uruguay.

SUPPLY PLANNING The supply plan must account for the procurement and supplier lead times
(SUGGESTED DELIVERY as well as the time required for country-specific importation processes;
FREQUENCY) in total this may require a lead time of 4-6 months. The time required
for in-country distribution must also be considered.
For planning of orders and shipments, the size of an order may include all
of the product needs estimated for a year, though with multiple shipments.
For QFT-Plus Blood Collection Tubes, given their remaining shelf life is short
(minimum of 10 months as per the GDF agreement with QIAGEN), shipments
may be required at least twice a year in order to prevent stock-outs.
For QFT-Plus ELISA kits, given the relatively long shelf life (up to 24 months),
shipments may be made annually.

20
QUALITY ASSURANCE, For the blood handling and immune stimulation phases, the Mitogen tube
CONTROL, serves as a positive control. A QFT-Plus Negative test must generate
AND ASSESSMENT a Mitogen minus Nil value of ≥0.5 IU/ml. A low response to Mitogen may
result from insufficient lymphocytes, reduced lymphocyte activity due
to improper specimen handling, incorrect filling or mixing of the Mitogen
tube, or inability of the patient’s lymphocytes to generate IFN-γ.
The accuracy of the test results is dependent on the generation of an
accurate standard curve. The reconstituted IFN-γ standard is used with each
ELISA to construct the standard curve.
→ For the ELISA to be valid:
• the mean OD value for Standard 1 must be ≥0.600,
• the %CV for Standard 1 and Standard 2 replicate OD values must be ≤15%,
• replicate OD values for Standard 3 and Standard 4 must not vary
by more than 0.040 optical density units from their mean,
• the correlation coefficient (r) calculated from the mean absorbance
values of the standards must be ≥0.98. 5) the mean OD value for the Zero
standard (Green Diluent) should be ≤ 0.150. If the mean OD value
is >0.150 the plate washing procedure should be investigated.
These control parameters are calculated and reported by the QFT-Plus Analysis
Software and must be reviewed before test results can be interpreted.
Each laboratory should determine appropriate types of control materials
to be used and the frequency of testing in accordance with applicable
accrediting organizations. For example, known positive/negative samples
or the Quantiferon Control Panel vials11 can be used to control performance
between runs and operators. Proficiency testing programs for IGRAs are also
available such as ones from the UK National External Quality Assessment
Service (UK NEQAS), INSTAND e.V., Society for Promoting Quality Assurance
in Medical Laboratories, and College of American Pathologists (CAP).
Key quality indicators that should be monitored monthly include the percentage
of runs with invalid standard curves; percentage of indeterminate, errors
or invalid results; percentage of positive results; distribution of IFN-γ
concentrations; and turnaround times (ANNEX 2). Targets should be set for all
indicators that are monitored, and any unexplained change in quality
indicators, such as an increase in error rates or a change in positivity rate
should be documented and investigated.

RECORDING Both the standard qualitative test interpretation (positive, negative,

AND REPORTING indeterminate), together with the criteria for test interpretation should be
reported.

11. https://www.qiagen.com/de/products/diagnostics-and-clinical-research/transplant/quantiferon-transplant/quantiferon-control-panel

21
 QUANTIFERON-TB® GOLD PLUS
(QFT-PLUS) IGRA

COST OF KIT AND COST PER PATIENT SAMPLE

(ELISA KITS AND BLOOD COLLECTION TUBES ARE REQUIRED.)

PATIENT COST PER

GDF ITEM COST OF KIT UNITS
PRODUCT SAMPLES PATIENT
NUMBER IN GDF CATALOG PER KIT
TESTED SAMPLE

QuantiFERON-TB Gold Plus 2 x 96 wells

106671 $279.84 44
(QFT-Plus) ELISA 2 x 96 + reagents

$6.36
QuantiFERON-TB Gold Plus
20 x 96 wells
(QFT-Plus) ELISA 20 x 96 106672 $2,798.40 440
+ reagents
(reference lab pack)

QFT-Plus Blood Collection

106673
200 Tubes
$477.00 200 tubes 50
QFT-Plus Blood Collection
106674
200 Tubes - High Altitude12
$9.54
QFT-Plus Blood Collection
106675
40 Tubes Single Patient

$95.40 40 tubes 10
QFT-Plus Blood Collection
40 Tubes Single Patient 106676
High Altitude

ORDER SIZES AND DELIVERY PLANS FOR TESTING 5,000, 20,000 OR 100,000 PEOPLE IN ONE YEAR
(ELISA KITS AND BLOOD COLLECTION TUBES ARE REQUIRED.)

GDF ITEM 5,000 20,000 100,000 SHELF NUMBER OF

PRODUCT
NUMBER TESTS TESTS TESTS LIFE DELIVERIES PER YEAR

QFT-Plus ELISA 2 x 96 106671 114 455 2,273

OR 24
1
QFT-Plus ELISA 20 x 96 months
106672 12 46 228
(reference lab pack)

QFT-Plus Blood Collection 106673

200 Tubes
OR 100 400 2,000
QFT-Plus Blood Collection 106674
200 Tubes - High Altitude
OR 6
4*
QFT-Plus Blood Collection 106675 months
40 Tubes Single Patient
OR
500 2,000 10,000
QFT-Plus Blood Collection
40 Tubes Single Patient 106676
High Altitude

*Second delivery after month 3, third after month 6, fourth after month 9.
12. Standard QFT-Plus Blood Collection Tubes can be used up to an altitude of 810 meters above sea level. High Altitude QFT-Plus Blood Collection Tubes can be used between 1,020 meters above sea level to an altitude of 1,875 meters
above sea level. J Infect, 2020. 80(5): p. 536-546. Doi: 10.1016/j.jinf.2020.02.009

22
2.
WANTAI TB-IGRA

24 PRINCIPLES OF THE ASSAY

24 PERFORMANCE
25 EQUIPMENT TO PERFORM THE ASSAY
26 HOW TO PERFORM THE ASSAY
28 OPERATIONAL CONSIDERATIONS

23
 WANTAI TB-IGRA

WANTAI TB-IGRA

The WANTAI TB-IGRA is operationally similar to the QFT-Plus

IGRA in that there is a blood collection and antigen
stimulation phase followed by detection of IFN-γ by ELISA.
Two key differences are 1) the WANTAI IGRA uses one tube
containing antigens for stimulation whereas the QFT-Plus IGRA
uses two tubes (TB1 and TB2) and 2) the stimulating antigens
used in the WANTAI TB-IGRA are recombinant- proteins
(ESAT6 [first 80 amino acids] and the full-length CFP-10)
whereas the stimulating antigens in the QFT-Plus IGRA
are mixtures of synthetic peptides (ESAT6 and CFP-10).
Also, IFN-γ concentrations are reported as pg/ml instead
of IU/ml.

PRINCIPLES OF THE ASSAY

The basic principles of the WANTAI TB-IGRA are also incubation.
similar to those of the QuantiFERON-TB assays in that The ‘T’ tube (testing culture tube) contains TB-specific
an ELISA is used to measure the amount of IFN-γ stimulating antigens (ESAT-6, CFP-10). These antigens
released following stimulation of whole blood samples are recombinant-produced proteins.
with M. tuberculosis-specific antigens. Briefly, whole
Following incubation at 37°C for 20 to 24 hours,
blood is collected into a blood collection tube with lithium
the samples are then centrifuged, the plasma is removed
heparin as an anticoagulant and 1 ml dispensed into
and the amount of IFN-γ (pg/ml) is measured by ELISA.
each of three tubes (‘N’, ‘T’, ‘P’).
A WANTAI TB-IGRA is considered positive if the
The ‘N’ tube adjusts for background (e.g. excessive levels concentration of IFN-γ (pg/ml) in the T tube minus
of circulating IFN-γ or presence of heterophile antibo- the concentration in the N tube is greater than or equal
dies). The IFN-γ level of the N tube is subtracted from the to 14 and is greater than or equal to the concentration
IFN-γ level for the T tube and P tube. in the N tube divided by 4 [i.e. T-N ≥14 and ≥ N/4].
The ‘P’ tube (positive control tube) is a positive control A low response to the positive control (P-N <20) indicates
for the ability of the lymphocytes to produce IFN-γ and an indeterminate result when a blood sample also has
also serves as a control for correct blood handling and a negative response to the TB antigens (FIGURE 6).

PERFORMANCE
Using the previously described approach relying the manufacturer compared the performance
on surrogate measures, the manufacturer showed of the WANTAI TB-IGRA with that of the QuantiFERON-TB
that the WANTAI TB-IGRA has a sensitivity of 97.1% Gold In-Tube assay and the T-Spot.TB assay.
(68/70 test subjects) for detecting persons known to have In two published studies, the agreement between the
a TB infection (i.e., persons with active TB disease) WANTAI TB-IGRA and the QFT-TB Gold In-Tube assay
and a specificity of 94.8% (199/210 test subjects) was good with a kappa statistic of 0.79 (95% CI: 0.60
for correctly identify persons thought not to have to 0.99).
a TB infection.13 In three published studies the agreement with T-Spot.
In addition to the use of surrogate measures, TB was very good with a kappa statistic of 0.87.13

13. WANTAI TB-IGRA brochure: https://www.ystwt.cn/wp-content/uploads/2018/04/Wantai-TB-IGRA.pdf

24
FIGURE 6

ALGORITHM FOR INTERPRETING No Is N ≤ 400?

RESULTS OF THE WANTAI TB-IGRA

Yes

No Is T-N ≥ 14?

Yes

N = concentration of IFN-γ (pg/ml)

in the Background Control Tube No Is P-N ≥ 20? No Is T-N ≥ N/4?
P = concentration of IFN-γ (pg/ml)
in the Positive Control Tube
Yes Yes*
T = concentration of IFN-γ (pg/ml)
in the Testing Tube
* = P-N any value Indeterminate Negative Positive

EQUIPMENT TO PERFORM THE ASSAY

MATERIALS REQUIRED BUT NOT PROVIDED MATERIALS PROVIDED

Phlebotomy materials include lithium heparin collection Microwell plate

tube
Background Control Culture Tube
Calibrated variable volume pipettes with disposable tips
Testing Culture Tube
Calibrated multichannel pipettes with disposable tips
Positive Control Culture Tube
Microplate shaker
Interferon gamma standard
Deionized or distilled water
HRP-conjugate
Microplate washer
Specimen diluent
Microplate reader fitted with 450 nm filter
and 620 nm to 650 nm reference filter Standard diluent

37°C ± 1°C incubator (CO2 not required) Wash buffer

Refrigerator 2°C-8°C Chromogen Solution A

Centrifuge for separation of plasma Chromogen Solution B

Disposable gloves Stop Solution

25
 WANTAI TB-IGRA

HOW TO PERFORM THE ASSAY

(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

PHASE 1 : BLOOD COLLECTION, ANTIGEN STIMULATION AND HARVESTING OF PLASMA

1 2 3 4
SPECIMEN DISPENSING CENTRIFUGATION AND
SPECIMEN COLLECTION AND MIXING INCUBATION HARVESTING PLASMA

Label each tube Label each tube Immediately after mixing, After incubation,
appropriately. appropriately. place the tubes in a centrifuge the specimens
37°C ± 1°C incubator. at 3000 to 5000 rpm for
For each patient, collect Gently invert the tubes 10 minutes to separate
4 ml of blood by 3-5 times to mix Incubate the tubes plasma and red blood
venipuncture directly into the specimens. in an upright position cells.
a blood collection tube for 20 to 24 hours.
with lithium heparin Dispense 1 ml of the whole Harvest the plasma using
as the anticoagulant. blood specimen into each a pipette.
Mix well. of the N, T and P tubes. The harvested plasma
The tube may be stored may be stored at 2°C
Gently invert the tubes
at 20°C to 27°C for up to 16 to 8°C for up to 2 days
5 times to mix
hours before the specimen or below -15°C for longer
the specimens.
dispensing step. times.
Store plasma in small
volume aliquots to avoid
multiple freeze-thaw
cycles. The manufacturer
recommends no more
than 2 freeze-thaw cycles.

26
HOW TO PERFORM THE ASSAY (CONTINUED)
(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

PHASE 2 : DETECTION OF IFN-γ AND GENERATION OF RESULTS

5 6 7

REAGENT PREPARATION PERFORM THE TB-IGRA ELISA CALCULATE RESULTS

Allow reagents and specimens Add 20μl of Specimen diluent to If the results are based on a single
to reach room temperature each well except the Blank well. filter plate reader, subtract the
(18°C to 30°C) for at least absorbance of the Blank well from
15-30 minutes. Add 50 μl of the standards and that of each test well. If a dual filter
50 μl of the specimen samples into (450nm/600-650nm) plate reader
Dilute the concentrated wash buffer their respective wells and mix is used, do not subtract
1:20 with distilled or deionized water. by gently tapping the plate. the absorbance of the Blank well
from that of the test wells.
Add distilled or deionized water Cover the plate with the plate cover
to the ampule according to and incubate for 60 minutes at 37°C. Construct a standard curve based on
the volume printed on the label the absorbance values of the wells
of the ampule to reconstitute Add 50 μl of the HRP-Conjugate
containing the various solutions
the freeze-dried standard. Reagent to each well except the Blank
of the standard and determine
This generates the 400pg/ml well and mix by gently tapping the plate.
the linear regression equation.
standard.
Cover the plate with the plate cover
Enter the absorbance values
Gently mix until it is homogeneously and incubate for 60 minutes at 37°C.
of the wells containing the plasma
dissolved. samples into the linear regression
At the end of the incubation, wash
each well 5 times with the diluted equation and calculate
Prepare a series of two-fold dilutions
wash buffer. Each time allow the the corresponding IFN-γ
using the Standard Diluent.
wells to soak for 30 to 60 seconds. concentration.
The final concentrations
of the standard samples should be After the final wash, turn the plate
400pg/ml, 200pg/ml, 100pg/ml, onto blotting paper and gently tap to
50pg/ml, 25pg/ml and 12.5pg/ml. remove any remaining wash buffer.

Set the strips needed in the strip Add 50 μl of Chromogen Solution A

holder. and 50 μl of Chromogen Solution B to
each well including the blank well and
Include 1 well for the plasma sample gently tap the plate to mix. Incubate the
of each culture tube, two wells plate at 37°C in the dark for 15 minutes.
for each standard, and one well
for a blank. Add 50 μl of Stop Solution to each
well and gently mix.

Calibrate the plate reader using the

Blank well and read the absorbance
of each well at 450 nm. (NB: If
the results will be determined by
using a dual wavelength plate
reader, the requirement for the
use of a Blank well could be
omitted. If a dual filter instrument
is used, set the dual-wavelength at
450nm/600~650nm). Absorbance
should be measured within 10
minutes of stopping the reaction.

27
 WANTAI TB-IGRA

FIGURE 7

WORKFLOW OF THE WANTAI TB

37°C

20-24 HOURS

Dispense
Collect blood Incubate Harvest Measure Calculate
specimen into
samples 20-24hr plasma IFN-γ (ELISA) results
tubes and mix

OPERATIONAL CONSIDERATIONS

CONSIDERATIONS Because of the need for sophisticated laboratory equipment and highly
FOR SITE SELECTION skilled laboratory personnel to perform the ELISA and interpret test results,
the test is best suited for implementation in intermediate or national
reference laboratories. Because of the use of ELISAs for other diseases,
national programs may be able to leverage collaboration with other,
non-TB-specific laboratories, having capacity for blood draws and ELISA
testing. The biosafety precautions for the WANTAI TB-IGRA are the same as
most ELISAs.
If the test is located in a centralized site, it will be necessary to establish
efficient specimen transport systems to ensure that blood samples can be
transported from peripheral sites to the IGRA testing laboratory within
the recommended time frames. For the WANTAI TB- IGRA, the maximum time
between blood collection and dispensing into the N, T, and P tubes
at the testing laboratory is 16 hours.

THROUGHPUT The time required to perform the WANTAI TB-IGRA test is estimated below;
AND TIME REQUIRED the time of testing multiple samples when batched is also indicated:
FOR PERFORMING ASSAY → 37°C incubation of blood tubes: 20 to 24 hours.
→ ELISA: Approx. 3 hours for one ELISA plate.
• An ELISA plate can contain up to 28 patient samples.
• <1 hour of labor.
• Add 10 to 15 minutes for each extra plate.

28
STORAGE → Kit reagents
AND HANDLING • Store kit reagents at 2°C to 8°C.
• Shelf life: 13 months (expiration date printed on each package).

→ Reconstituted and unused reagents

• Once opened, unused microwell strips may be stored at 2°C to 8°C
in a plastic storage bag (provided) with desiccant for up to 3 weeks.
• Once reconstituted, the standards should be used on the same day.
• Once opened, the HRP-Conjugate is stable for 3 weeks at 2°C to 8°C.
• Working strength wash buffer may be stored at room temperature for up
to 1 week or 2 weeks at 2°C to 8°C.
• Once opened, the Specimen Diluent is stable for 3 weeks at 2°C to 8°C.
• Once opened, the Standard Diluent is stable for 3 weeks at 2°C to 8°C.
• Once opened, the Chromogen Solution A is stable for 3 weeks at 2°C to 8°C.
• Once opened, the Chromogen Solution B is stable for 3 weeks at 2°C to 8°C.
• Once opened, the Stop Solution is stable for 3 weeks at 2°C to 8°C.

INTERPRETATION OF RESULTS → Result interpretation

AND QUALITY CONTROL If the result reading is based on a single filter plate reader, the results should
be calculated by subtracting the “A” value of the Blank well from the print
report values of specimens and controls. For dual filter plate readers,
do not subtract the A value of the Blank well from the print report values
of specimens and controls. The limit of the blank (LOB) should not be higher
than 3pg/ml. The linear range for the assay is 12.5pg/ml-400pg/ml.
Should the IFN-γ concentration of a specimen be >400pg/ml; the specimen
should be diluted using the standard diluent and the test repeated.

→ Quality control
For the blood handling and immune stimulation phases, the Positive Control
tube (’T’) serves as a positive control. The result should be considered invalid
if either the correlation coefficient of the dose-response curve Correlation
coefficient, r>0.9900, or the mean of A values of 400pg/ml Standard<1.0.
Samples with an invalid result should be repeated. A low response of positive
control may result from insufficient lymphocytes, reduced lymphocyte activity
due to improper specimen handling, incorrect filling or mixing of the Positive
Control Mitogen tube, or inability of the patient’s lymphocytes to generate
IFN-γ.
Important internal controls that are conducted with each ELISA are the known
samples of IFN-γ used to construct the standard curve.
→ For the ELISA to be valid:
• the mean absorbance value for the 400pg/ml standards must be ≥1.0,
• the correlation coefficient (r) calculated from the mean absorbance values
of the standards must be ≥0.99.
Proficiency testing programs for IGRAs are available such as ones from the
UK National External Quality Assessment Service (UK NEQAS), INSTAND e.V.,
Society for Promoting Quality Assurance in Medical Laboratories, and College
of American Pathologists (CAP).
Key quality indicators that should be monitored monthly include the percentage
of runs with invalid standard curves; percentage of indeterminate, errors
or invalid results; percentage of positive results; distribution of IFN-γ
concentrations; and turnaround times (ANNEX 2). Targets should be set for all
indicators that are monitored, and any unexplained change in quality
indicators, such as an increase in error rates or a change in positivity rate
should be documented and investigated.

29
 WANTAI TB-IGRA

INTERPRETATION OF THE RESULTS

N P-N T-N RESULT INTERPRETATION

Infected
N
any value ≥14 and ≥ — postive with Mycobacterium tuberculosis
4
(active, latent or inapparent infection

≥20 <14 negative

Not infected
≤400
N with Mycobacterium tuberculosis
≥20 ≥14 but < — negative
4

<20 <14 indeterminate

Cannot determine
<20 ≥14 but < indeterminate whether Mycobacterium tuberculosis
infection
≤400 any value any value indeterminate

The concentration of Testing Culture Tube (T)=T, the concentration of Background control tube (N)=N, and the concentration of Positive control tube (P) =
P(Unit=p/ml). The Background Control Tube (‘N’) assesses the level of circulating IFN-γ or presence of heterophile antibodies. A valid test must generate
an ‘N’ value of ≤400pg/ml.

RECORDING Both the standard qualitative test interpretation (positive, negative,

AND REPORTING indeterminate) and the quantitative assay measurements should be
reported, together with the criteria for test interpretation.

30
3.
STANDARDTM E
TB-FERON IGRA

32 PRINCIPLES OF THE ASSAY

33 PERFORMANCE
35 EQUIPMENT TO PERFORM THE ASSAY
36 HOW TO PERFORM THE ASSAY
38 OPERATIONAL CONSIDERATIONS

31
 STANDARDTM E TB-FERON IGRA

STANDARDTM E TB-FERON IGRA

The SD Biosensor TBF assay is operationally similar to the

QFT-Plus IGRA in that there is a blood collection and antigen
stimulation phase followed by detection of IFN-γ by ELISA.
Two key differences are 1) the QFT-Plus IGRA uses two tubes
(TB1 and TB2) whereas the TBF assay uses one tube
containing antigen for stimulation and 2) the stimulating
antigens in the QFT-Plus IGRA are mixtures of synthetic
peptides (ESAT6 and CFP-10) whereas the stimulating antigens
used in the TBF assay are recombinant-produced full-length
proteins (ESAT6, CFP-10, and TB7.7).

PRINCIPLES OF THE ASSAY

For the TBF IGRA, whole blood specimens are but thoroughly mixed with the collected blood to ensure
collected into each of the three TBF tubes. The tubes that the antigens on the tube walls are completely
differ by which antigens or controls have been dried dissolved. The tubes are then incubated in an upright
onto the walls of the tube. Alternatively, blood may be position at 37°C for 16 to 24 hours,
collected in a single generic BCT that contains lithium during which time the immune stimulation occurs.
heparin as the anticoagulant and then transferred to TBF The samples are then centrifuged, the plasma is removed
tubes. and the amount of IFN-γ (IU/ml) is measured by ELISA.
The Nil tube adjusts for background (e.g. excessive levels
of circulating IFN-γ or presence of heterophile A TBF assay is considered positive if the IFN-γ response
antibodies). The IFN-γ level of the Nil tube is subtracted to the TB Antigen tube is significantly above the Nil IFN-γ
from the IFN-γ level for the TB Antigen tubes and Mitogen IU/ml value. The plasma sample from the Mitogen tube
tubes. serves as an IFN-γ positive control for each specimen
tested. A low response to Mitogen (<0.5 IU/ml) indicates
The Mitogen tube is a positive control for the ability
an indeterminate result when a blood sample also has
of the lymphocytes to produce IFN-γ and also serves
a negative response to the TB antigens.
as a control for correct blood handling and incubation.
The TB antigen tube contains full-length The manufacturer also describes the option of using only
recombinant-produced ESAT-6, CFP-10, and TB7.7 the Nil and TB antigen tubes. The testing protocol
proteins. is the same (minus the use of the Mitogen tube). The
The contents of the inoculated TBF tubes must be gently, interpretation of the results follows a similar algorithm.

32
PERFORMANCE
Performance was assessed in a study of 705 patients14 could thus be suggested to be extrapolated to the TBF
by comparing the results obtained using the TBF IGRA IGRA.
with the results obtained with the QFT-Plus assay.
The overall positive agreement was 96.7% (119/123), Information on the performance of the two-tube assay
the overall negative agreement was 95% (553/582) (Nil and TB antigen tubes only) is lacking. In particular,
and the overall agreement was 95.3% (672/705). it is unknown how the lack of a mitogen control in the
two-tube assay affects the comparison with the QFT-Plus
The performance characteristics of the QFT-Plus assay test, particularly in immunocompromised individuals.

FREQUENCY % (95% CI)

Negative Agreement 672/705 95.3% (93.5–96.8%)

Positive Agreement 119/123 96.7% (91.9–99.1%)

Overall Agreement 197/206 95.6% (92.9–98%)

TBF IGRA QFT-PLUS RESULTS

RESULTS
Negative Positive Total

Negative 553 4 557

Positive 29 119 148

Total 582 123 705

14. Standard E TB Feron ELISA kit insert.

33
 STANDARDTM E TB-FERON IGRA

FIGURE 8

ALGORITHM FOR INTERPRETING TB-Nil ≥0.35

No
RESULTS OF THE STANDARD U/ml
E TB-FERON IGRA (3 TUBE ASSAY)

Yes

TB-Nil ≥25% of
No
Nil IU/ml value

Yes

Mitogen-Nil
<0.50 IU/ml
Yes Indeterminate No Nil ≤8.0 IU/ml
and/or
Nil >8.0 IU/ml

Yes
No

Negative Positive

FIGURE 9

ALGORITHM FOR INTERPRETING TB-Nil ≥0.35

No
RESULTS OF THE STANDARD U/ml
E TB-FERON IGRA (2 TUBE ASSAY)

Yes

TB-Nil ≥25% of
No
Nil IU/ml value

Yes

Nil ≤8.0 IU/ml Yes Indeterminate No Nil ≤8.0 IU/ml

No Yes

Negative Positive

34
EQUIPMENT TO PERFORM THE ASSAY

MATERIALS REQUIRED BUT NOT PROVIDE MATERIALS PROVIDED

Phlebotomy materials TB-Feron Nil Tubes (gray cap)

Calibrated variable volume pipettes with disposable tips TB-Feron TB Antigen Tubes (red cap)

Calibrated multichannel pipettes with disposable tips TB-Feron Mitogen Tubes (purple cap)

Microplate shaker Standard E TB-Feron ELISA ki

Deionized or distilled water

Microplate reader fitted with 450 nm filter

and 620 nm to 650 nm reference filter

37°C ± 1°C incubator (CO2 not required)

Refrigerator 2°C-8°C

Centrifuge for separation of plasma

Disposable gloves

FIGURE 10

WORKFLOW OF THE STANDARD E TB-FERON IGRA

37°C

16-24 HOURS

Collect (or dispense) blood samples Incubate Harvest Measure Calculate

into TB-Feron tubes and mix 16-24hr plasma IFN-γ (ELISA) results

35
 STANDARDTM E TB-FERON IGRA

HOW TO PERFORM THE ASSAY

(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

PHASE 1 : BLOOD COLLECTION, ANTIGEN STIMULATION AND HARVESTING OF PLASMA

1 2 3
INCUBATION AND HARVESTING
SPECIMEN COLLECTION MIXING OF TUBE CONTENTS OF PLASMA

Label each tube appropriately. Immediately after filling the Incubate the TB-Feron Tubes upright
Allow tubes to come to room TB-Feron Tubes, shake them at 37°C ± 1°C for 16 to 24 hours.
temperature (15-25°C). ten (10) times just firmly enough The incubator does not require CO2
to make sure the entire inner surface or humidification.
For each patient, collect 1 ml of blood of the tube is coated with blood. This
by venipuncture directly into each of will dissolve antigens on tube walls. Harvesting of plasma is facilitated
the TB-Feron tubes. This procedure Do not shake excessively. by centrifuging the tubes at
should be performed by a trained 2200–2300 x g (RCF) for 15 minutes.
phlebotomist or equivalent. Transfer the TB-Feron Tubes to
a 37°C ± 1°C incubator as soon as Harvest plasma samples using a
Alternatively, draw blood into possible and no more than pipette.
a lithium-heparin tube and dispense 16 hours after blood collection. Avoid pipetting up and down or
1 ml into each of the three TB-Feron Prior to incubation at 37°C ± 1°, mixing the plasma by any means
tubes. If necessary, blood may be maintain the tubes at room prior to harvesting.
stored in the lithium-heparin tube at temperature (20°C ± 5°C). At all times, take care not to disturb
15°C to 25°C for up to 16 hours before The total time from blood the material on the surface of the
dispensing into the TB-Feron tubes. collection to initiating the 37°C gel.
The total time from blood incubation should not exceed 16 hr. Separated plasma can be stored
collection to initiating the 37°C for up to one week at 2°C to 8°C.
incubation should not exceed Separated plasma samples can also
16 hours. be stored below –20°C for extended
periods.

36
HOW TO PERFORM THE ASSAY (CONTINUED)
(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

PHASE 2 : DETECTION OF IFN-γ AND GENERATION OF RESULTS

4 5 6
PERFORM STANDARD
PREPARE IFN-γ STANDARDS E TB-FERON ELISA CALCULATE RESULTS

Label S1, S2, S3, and S4 on 4 empty Add 50μl of prepared Working Calculate results using the
tubes. Detector solution into each STANDARD E ANALYSIS SOFTWARE.
of the wells These calculations can also be
Add 300μl of ELISA Diluent to each performed using software available
tube. Add 50μl of STANDARD 1 to 4 and with microplate readers, standard
the specimens into the appropriate spreadsheets, and statistical
Add 100μl of Reconstituted wells of the antibody-coated software (e.g. Microsoft Excel).
STANDARDs to STANDARD tube 1 (S1) microplate. It is recommended that these
and mix thoroughly
packages be used to calculate the
(S1 contains 4 IU/ml). Mix gently by tapping the pate.
regression analysis, the coefficient
Transfer 100μl of the STANDARD Cover the plate with the attached of variation (%CV) for the standards,
tube1 (S1) solution to STANDARD tube plate sealer and incubate at 37±1°C and the correlation coefficient (r)
2 (S2) for 1 hour. of the STANDARD curve.
(S2 contains 1 IU/ml).
Wash the wells five times with 350μl
Transfer 100μl of the STANDARD of diluted wash buffer and aspirate
tube2 (S2) solution to STANDARD all liquid from the wells. Or, wash
tube 3 (S3) the wells using an automatic washer
with 350μl of diluted wash buffer.
ELISA Diluent serves as a zero
STANDARD (S4). Add 100μl of TMB substrate into each
of the wells.

Incubate for 30 minutes at room

temperature (15-25°C) in the dark.

Add 100μl of stop solution into each

of the wells. Mix by gentle shaking.

Read the absorbance at 450nm

in an ELISA plate reader
(with reference wavelength between
620 nm and 650 nm) within 30
minutes.

37
 STANDARDTM E TB-FERON IGRA

OPERATIONAL CONSIDERATIONS

CONSIDERATIONS Because of the need for sophisticated laboratory equipment and highly
FOR SITE SELECTION skilled laboratory personnel to perform the ELISA and interpret test results,
the test is best suited for implementation in intermediate or national
reference laboratories. Because of the use of ELISAs for other diseases,
national programs may be able to leverage collaboration with other,
non-TB-specific laboratories, having the capacity for blood draws and ELISA
testing. The biosafety precautions for the TBF test are the same as most tests
that require blood draws and ELISAs.
If the test is located in a centralized site, it will be necessary to establish
efficient specimen transport systems to ensure that blood samples can be
transported from peripheral sites to the IGRA testing laboratory within
the recommended time frames. For the TBF test, the maximum time between
blood collection in lithium-heparin tubes and processing at the testing
laboratory is 16 hours.

THROUGHPUT The time required to perform the TB-Feron IGRA test is estimated below;
AND TIME REQUIRED the time of testing multiple samples when batched is also indicated:
FOR PERFORMING ASSAY → 37°C incubation of blood tubes: 16 to 24 hours.
→ ELISA: Approx. 2.5 hours for one ELISA plate.
• A 96-well ELISA plate can contain samples from up to 28 individuals
(12 wells used for IFN-γ standards).
• <1 hour of labor.
• Add 10 to 15 minutes for each extra plate.

STORAGE → TB-Feron tubes

AND HANDLING • Store TB-Feron tubes at 2°C to 25°C.
• Shelf life: 15 months (expiration date printed in the package
and in the label of each tube).

→ Kit reagents
• Store kit reagents at 2°C to 8°C.
• Always protect Enzyme Substrate Solution from direct sunlight.
• Shelf life: 15 months (expiration date printed in the package
and in the label of each tube).
• Store STANDARD E TB-Feron Control at 2°C to 30°C.

→ Reconstituted and unused reagents

• The diluted wash buffer may be kept for up to 1 week if stored
at 15°C to 25°C.
• The working detector solution can be stored for up to 4 hours
at 2°C to 8°C.

38
ORDER PLANNING

PRODUCT SPECIMEN PACK SIZE CAT NO.

ETB-Feron Elisa (2 plates) Plasma 192 wells/kit 07TBF10C

ETB-Feron Elisa Tube 100 WB Mitogen tube x 100 07TBFA10

WB TB Antigen tube x 100

ETB-Feron Elisa Tube 200 07TBFA20
WB Nile tube x 100

ETB-Feron Elisa Control Lv1 x 15 / Lv2 x 15 / Lv3 x 15 07TBFC10

QUALITY ASSURANCE, For the blood handling and immune stimulation phases, the Mitogen tube
CONTROL, serves as a positive control. A valid test must generate a Mitogen minus Nil
AND ASSESSMENT value of ≥0.5 IU/ml. Low response to Mitogen may result from insufficient
lymphocytes, reduced lymphocyte activity due to improper specimen
handling, incorrect filling or mixing of the Mitogen tube, or inability
of the patient’s lymphocytes to generate IFN-γ.
Important internal controls that are conducted with each ELISA are the known
samples of IFN-γ used to construct the standard curve.
→ For the ELISA to be valid:
• the mean OD value for Standard 1 must be ≥0.600,
• the %CV for Standard 1 and Standard 2 replicate OD values must be ≤15%,
• replicate OD values for Standard 3 and Standard 4 must not vary
by more than 0.040 optical density units from their mean,
• the mean O.D value for S4 must be 0.150 or less,
• the correlation coefficient (r) calculated from the mean absorbance values
of the standards must be ≥0.98.
Proficiency testing programs for IGRAs are available such as ones from
the UK National External Quality Assessment Service (UK NEQAS), INSTAND
e.V., Society for Promoting Quality Assurance in Medical Laboratories,
and College of American Pathologists (CAP).
Key quality indicators that should be monitored monthly include the
percentage of runs with invalid standard curves; percentage of indeterminate,
errors or invalid results; percentage of positive results; distribution of IFN-γ
concentrations; and turnaround times (ANNEX 2). Targets should be set for
all indicators that are monitored, and any unexplained change in quality
indicators, such as an increase in error rates or a change in positivity rate
should be documented and investigated.

RECORDING Both the standard qualitative test interpretation (positive, negative,

AND REPORTING indeterminate) and the quantitative assay measurements should be
reported, together with the criteria for test interpretation.

39
 4.
T-SPOT®.TB ASSAY

41 PRINCIPLES OF THE ASSAY

41 PERFORMANCE
43 EQUIPMENT TO PERFORM THE ASSAY
44 HOW TO PERFORM THE ASSAY
46 OPERATIONAL CONSIDERATIONS

40
 T-SPOT®.TB ASSAY

T-SPOT®.TB ASSAY

The T-SPOT®.TB test is an in vitro diagnostic test

for the detection of effector T cells that respond to stimulation
by M. tuberculosis antigens ESAT-6 and CFP-10 by detecting
the IFN-γ secreted in the vicinity of T cells by capturing
the IFN-γ on an antibody-coated membrane.

PRINCIPLES OF THE ASSAY

The immune response to infection with M. tuberculosis sequences of the entire amino acid sequence
is mediated predominantly through T cell activation. of ESAT-6 (Panel A).
As part of this response, T cells are sensitized A mixture of peptides representing overlapping
to M. tuberculosis antigens and the activated effector sequences of the entire amino acid sequence
T cells, both CD4+ and CD8+, produce IFN-γ when of CFP-10 (Panel B).
stimulated by these antigens. The T-SPOT.TB test uses
the enzyme-linked immunospot (ELISPOT) method The PBMCs are incubated for 16 to 20 hours with
to enumerate M. tuberculosis-sensitized T cells the antigens to allow the stimulation of any sensitized
by capturing IFN-γ in the vicinity of the T cells from which T cells present. Any secreted IFN-γ is captured by specific
it was secreted. antibodies on the surface of the membrane, which forms
the base of the well. After incubation, the cells and other
Peripheral blood mononuclear cells (PBMCs) are unwanted materials are removed by washing.
separated from a whole blood sample, washed, A second antibody, conjugated to alkaline phosphatase
counted, and then placed into microtiter wells where and directed to a different epitope on the IFN-γ molecule,
they are exposed to: is added and binds to the IFN-γ captured on the
membrane surface. Any unbound conjugate is removed
A nil control which adjusts for background
by washing. A soluble substrate is added to each well;
IFN-γ-producing PMBCs. The number of spots
this is cleaved by bound enzymes to form a (dark blue)
in the nil control is subtracted from the number of spots
spot of insoluble precipitate at the site of the reaction.
in the positive control well and antigen wells.
A positive control (phytohemagglutinin) which assesses Evaluating the number of spots obtained provides
cell functionality. a measurement of the abundance of M. tuberculosis
A mixture of peptides representing overlapping sensitive effector T cells in the peripheral blood.

PERFORMANCE
Using the previously described approach relying to have a TB infection (i.e. persons with active TB
on surrogate measures, the manufacturer showed disease) and a specificity of 97.1% (297/306 test subjects)
that the T-SPOT.TB test has a sensitivity of 95.6% for correctly identifying persons thought not to have
(175/183 test subjects) for detecting persons known a TB infection.

41
 T-SPOT®.TB ASSAY

FIGURE 11

ALGORITHM FOR INTERPRETING RESULTS OF THE T-SPOT.TB ASSAY

Nil Control Count

Nil
Positive ≤10 spots >10 spots
Panel A
Panel B
Inavlid

Positive Control

≥20 spots <20 spots

Panel A-Nil ≥8 spots Panel A-Nil ≥8 spots

Positive
or Panel B-Nil ≥8 spots or Panel B-Nil ≥8 spots

The highest of Panel The highest of Panel

A-Nil or Panel B-Nil Borderline A-Nil or Panel B-Nil
is 5, 6 or 7 spots is 5, 6 or 7 spots

Both Panel A-Nil Both Panel A-Nil

Negative Invalid
and Panel B ≤ 4 spots and Panel B ≤ 4 spots

SOURCE: REPRODUCED FROM T-SPOT.TB PACKAGE INSERT

FIGURE 12

WORKFLOW OF THE T-SPOTPOT.TB TEST

PBMCs

PRE-COATED WELLS
Wash, develop
Collect Remove PBMs, Add PBMs and antigens
and dry plate,
peripheral Centrifuge wash to 4 wells,
and count the coloured
venous blood and count and incubate overnight
spots in each well

SOURCE: REPRODUCED FROM T-SPOT.TB PACKAGE INSERT

42
EQUIPMENT TO PERFORM THE ASSAY

MATERIALS REQUIRED BUT NOT PROVIDED MATERIALS PROVIDED

Phlebotomy materials Microtiter plate: supplied as 12x 8-well strips in a frame

or as a solid 96-well plate, coated with a mouse
Blood collection tubes, such as Vacutainer® CPT™
or heparinized tubes Mmonoclonal antibody to IFN-γ

Class II biosafety cabinet (recommended) Panel A: contains ESAT-6 antigens, bovine serum
albumin, and antimicrobial agents
T-Cell Xtend® reagent (if used)
Panel B: contains CFP10 antigens, bovine serum albumin
and antimicrobial agents
Ficoll® and 15 mL centrifuge tubes (if not using CPT tubes)

Positive Control: contains phytohemagglutinin (PHA),

Centrifuge for preparation of PBMCs
for use as a cell functionality control, bovine serum
(capable of at least 1800 RCF (g)
albumin and antimicrobial agents
and able to maintain the samples at 18°C -25°C)

200x concentrated Conjugate Reagent: mouse

Equipment and reagents to enable counting of PBMCs:
monoclonal antibody to the cytokine IFN-γ conjugated
e.g., a hemocytometer on a microscope or a suitable
to alkaline phosphatase
hematology analyzer

Substrate Solution: ready-to-use BCIP/NBTplus solution

Magnetic bead-based processing system, if T-Cell
Select™ is used instead of Leucosep™ tubes

Humidified incubator capable of 37 ± 1°C

with a 5% CO2 supply

Sterile cell culture medium such as GIBCO AIM-VTM

Adjustable pipettes and sterile pipette tips

8-well strip plate frame

Automatic microtiter plate washer or an 8 channel

or stepper pipette to manually wash plates

Sterile PBS solution

Distilled or deionized water

A means of visualizing the wells, or capturing a digital

image of the well, such as a stereomicroscope,
magnifying glass or plate imager to allow counting
of spots

43
 T-SPOT®.TB ASSAY

HOW TO PERFORM THE ASSAY

(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

1 2
PLATE SET UP AND INCUBATION
THE T-SPOT.TB TEST REQUIRES FOUR WELLS TO BE
SAMPLE COLLECTION AND PREPARATION USED FOR EACH PATIENT SAMPLE

Collect a blood sample according to the instructions Remove the pre-coated 8-well strips
supplied with the collection device. Store collected blood from the packaging, clip them into a plate frame
at room temperature (18°C–25°C) or at 10°C–25°C and allow them to equilibrate to room temperature.
if the T-Cell Xtend reagent is to be used. Or remove a solid 96-well plate from the packaging
Do not refrigerate or freeze. and allow it to equilibrate to room temperature.

Harvest PBMCs: Each patient sample requires the use of 4 individual

• When using CPT blood collection tubes, follow wells:
the manufacturer’s instructions for the separation • Add 50μl AIM V culture medium to each Nil Control
of PBMCs. well.
• When using blood collection vacutainers containing • Add 50μl Panel A solution to each well required.
heparin or citrate, separate PBMCs by centrifugation • Add 50μl Panel B solution to each well required.
through Ficoll-Paque Plus using published procedures. • Add 50μL Positive Control solution to each cell
• If Leucosep tubes, or the T-Cell Xtend reagent functionality control well.
(available from Oxford Immunotec) are used, follow the
protocols provided with these reagents. To each of the 4 wells to be used for a patient sample,
add 100μL of the patient’s final cell suspension
Collect the white, cloudy band of PBMCs using a pipette (containing 250,000 viable cells).
and transfer it to a 15ml conical centrifuge tube.
Make up the volume to 10ml with the cell culture medium. Incubate the plate in a humidified incubator at 37°C
Alternatively, a cell washing centrifuge, with 5% CO2 for 16–20 hours.
e.g., DiaCent-CW (Bio-Rad), may be used to facilitate
the cell washing stages. If this system is used then DPBS
should be used to wash the cells.

Centrifuge at 600xg for 7 minutes.

Pour off the supernatant and resuspend the pellet
in 1ml of the medium.

Make up the volume to 10mL with fresh medium

and centrifuge at 350xg for 7 minutes.

Pour off the supernatant and resuspend the pellet

in 0.7ml of AIM V culture medium.

Cell Counting and Dilution: The T-SPOT.TB test requires

2.5x105 viable PBMCs per well.
A total of four wells are required for each patient sample.
Perform a viable cell count, e.g. manual counting using
Trypan Blue and a haemocytometer or automated
counting using an appropriate instrument.
Calculate the concentration of viable cells present
in the stock cell suspension.
Prepare 500μL of the final cell suspension
at a concentration of 2.5x105 cells/100μl.
Ensure cells are thoroughly mixed before removing
an aliquot for dilution.

44
HOW TO PERFORM THE ASSAY (CONTINUED)
(ADAPTED FROM MANUFACTURER’S INSTRUCTIONS FOR USE)

3 4

SPOT DEVELOPMENT AND COUNTING RESULTS INTERPRETATION AND TEST CRITERIA

Remove the plate from the incubator. A Nil Control spot count in excess of 10 spots should be
considered as ‘Indeterminate’.
Discard the cell culture medium and add 200μl D-PBS Another sample should be collected from the individual
solution to each well. and tested.
Discard the D-PBS solution. A Positive Control spot count of <20 spots should be
Repeat the well washing a further 3 times with fresh considered as ‘Indeterminate’ unless either Panel A or
D-PBS solution for each wash. Panel B is ‘Positive’ as described in the next step, in which
case the result is valid.
Dilute concentrated Conjugate Reagent 200-fold in PBS
to create the working strength solution. The test result is ‘Positive’ if (Panel A minus Nil Control)
and/or (Panel B minus Nil Control) ≥ 6 spots.
Add 50μl working strength Conjugate Reagent solution
to each well and incubate at 2°C–8°C for 1 hour. The test result is ‘Negative’ if both (Panel A minus Nil
Control) and (Panel B minus Nil Control) ≤ 5 spots.
Discard the conjugate and perform four D-PBS washes
This includes values less than zero.
as described in the 2nd and 3rd steps above.
NB: If using the US version of the kit:
Add 50μl Substrate Solution to each well and incubate
• The test result is ‘Positive’ if (Panel A minus Nil
at room temperature for 7 minutes.
Control) and/or (Panel B minus Nil Control)
Wash the plate thoroughly with distilled or deionised ≥ 8 spots.
water to stop the detection reaction. • The test result is ‘Negative’ if both (Panel A minus Nil
Control) and (Panel B minus Nil Control) ≤ 4 spots.
Allow the plate to dry by standing it in a well-ventilated
area or in an oven at up to 37°C. Results where the highest of the Panel A or Panel B spot
count is such that the (Panel minus Nil) spot count is
Count and record the number of distinct, dark blue spots 5, 6, or 7 spots can be considered Borderline (equivocal),
on the membrane of each well. and retesting by collecting another patient sample
is recommended.
NB: a borderline result is a valid, reportable result.

45
 T-SPOT®.TB ASSAY

OPERATIONAL CONSIDERATIONS

CONSIDERATIONS Because of the need for highly skilled laboratory personnel to perform and
FOR SITE SELECTION interpret the T-SPOT.TB test, it is best suited for implementation in intermediate
or national reference laboratories. The biosafety precautions for the T-SPOT.
TB tests are the same as most tests that require BSL-2 level laboratories.
If the test is located in a centralized site, it will be necessary to establish
efficient specimen transport systems to ensure that blood samples can be
transported from peripheral sites to the T-SPOT.TB testing laboratory within
the recommended time frames. Blood samples must be stored at room
temperature and tested within 8 hours of blood collection, or within 32 hours
with storage at 10°C–25°C if the sample is treated with a means of removing
granulocytes, such as the use of the T-Cell Xtend reagent. Using the T-Cell
Select reagent extends the time from blood collection to processing to 54 hours.

TIME REQUIRED • Blood collection and sample processing: 3 hours.

FOR PERFORMING ASSAY A 96-well plate can accommodate samples from up to 24 individuals.
• 37°C incubation of blood tubes: 16 to 20 hours.
• Wash, develop, and dry plate and count spots: 1.5 to 2 hours.

STORAGE → Kit reagents

AND HANDLING • Store unopened kit reagents at 2°C to 8°C.
• Always protect Enzyme Substrate Solution from direct sunlight.
• The T-SPOT.TB test kit has a minimum remaining shelf life of 14 months
as per the Stop TB GDF agreement with Oxford Immunotec.
Always use before the expiration date printed on the kit label.
→ Reconstituted and unused reagents
• Store opened kit components at 2°C to 8°C Components must be used
within 8 weeks of opening.
• Working strength conjugate may be stored for up to six weeks at 2°C to 8°C
prior to use.

FORECASTING Eligible countries for T-SPOT.TB concessional prices through GDF include:
AND ORDER PLANNING → All low- and middle-income countries except:
Azerbaijan, Brazil, Bulgaria, China, Democratic People’s Republic of
Korea, India, Indonesia, Iran, Jordan, Kazakhstan, Lebanon, Malaysia,
Maldives, Morocco, Myanmar, Peru, Philippines, Russian Federation,
Thailand, Turkey, Tunisia, Ukraine, Vietnam.
→ Listed high-income countries:
Antigua and Barbuda, Bahamas, Barbados, Brunei Darussalam, New
Caledonia, Palau, Seychelles, St. Kitts and Nevis, Trinidad and Tobago.
To perform T-SPOT.TB IGRAs, products from the T-SPOT.TB test kit and AIM-V
culture medium are required. Furthermore, either Leucosep tubes or T-Cell
Select (plus generic blood collection tubes) are required. T-Cell Xtend, allowing
blood samples to be processed up to 32 hours after venipuncture, is optional.
The concessional prices reflect significant discounts from market pricing
in high-income countries. The least expensive option to perform T-SPOT.TB
IGRAs is when Leucosep tubes are used and T-Cell Xtend is not used; this
results in a cost per patient sample of $10.32. If T-Cell Xtend is used, the cost
per patient sample is $13.20. If T-Cell Select is used instead of Leucosep
tubes, the cost per patient sample is $13.35. The above costs per patient
sample do not take into consideration the costs of ancillary consumables.

46
 PATIENT COST PER
GDF ITEM COST OF KIT IN UNITS
PRODUCT SAMPLES PATIENT
NUMBER GDF CATALOG PER KIT
TESTED SAMPLE
T-SPOT.TB test kit 106665 $168.00 1 x 96 wells 24 $7.00

T-Cell Xtend15 106666 $115.00 3 x 2 ml 40 $2.88

T-Cell Select16 106667 $693.00 1 kit 144 $4.81

Leucosep tubes17 106668 $89.00 50 tubes 50 $1.78

AIM-V culture medium18 (50 mL) 106669 $23.00 50 ml 2.4 N/A

AIM-V culture medium (500 mL) 106670 $37.00 500 ml 24 $1.54

15. T-Cell Xtend allows for the sample storage time to be extended from 8 to 32 hours.
16. T-Cell Select reagent is used for isolation of peripheral blood mononuclear cells from whole blood.
17. Leucosep blood collection tubes facilitate separation of peripheral blood mononuclear cells from whole blood.
18. AIM-V sterile cell culture medium is used for the incubation step.

SUPPLY PLANNING The supply plan must account for the procurement and supplier lead times
(SUGGESTED DELIVERY as well as the time required for country-specific importation processes;
FREQUENCY) in total this may entail 4-6 months. The time required for in-country
distribution must also be considered.
For the planning of orders and shipments, the size of an order may include all
of the product needs estimated for a year, though with multiple shipments.
For the AIM-V culture medium, given the maximum shelf life of 9 months,
shipments are required at least 2-3 times a year in order to prevent stock-outs.
For T-Cell Select and T-SPOT.TB test kits, given the maximum shelf lives of
12 and 14 months, respectively, shipments may also be required twice a year.
For the remaining products, including Leucosep tubes and T-Cell Xtend,
given the maximum shelf life of 18 months and 38 months, respectively,
shipments may be made annually.

QUALITY ASSURANCE, For the blood handling and immune stimulation phases, the Positive Control
CONTROL, sample tube serves to assess cell functionality. A valid test must generate
AND ASSESSMENT a Positive Control spot count of ≥20 spots. A Positive Control spot count
of <20 spots should be considered as ‘Indeterminate’ unless either Panel A
or Panel B is ‘Positive’ as described above, in which case the result is valid.
A low response to the Positive Control may result from insufficient
lymphocytes, reduced lymphocyte activity due to improper specimen
handling, or the inability of the patient’s lymphocytes to generate IFN-γ.
Proficiency testing programs for IGRAs are available such as ones from the
UK National External Quality Assessment Service (UK NEQAS), INSTAND e.V.,
Society for Promoting Quality Assurance in Medical Laboratories and College
of American Pathologists (CAP).
Key quality indicators that should be monitored monthly include
the percentage of runs with invalid standard curves; percentage
of indeterminate, errors or invalid results; percentage of positive results;
distribution of IFN-γ concentrations; and turnaround times (ANNEX 2). Targets
should be set for all indicators that are monitored, and any unexplained
change in quality indicators, such as increase in error rates or a change
in positivity rate should be documented and investigated.

RECORDING Both the standard qualitative test interpretation (positive, negative,

AND REPORTING borderline, invalid) and the quantitative assay measurements should be
reported, together with the criteria for test interpretation.

47
 SUMMARY TABLE:
COMPARISON OF IGRAS
FOR THE DETECTION OF TB INFECTION

SUMMARY TABLE:
COMPARISON OF IGRAS FOR THE DETECTION OF TB INFECTION

QFT-PLUS WANTAI STANDARD E TBF T-SPOT.TB

Number of tubes used 4 (Nil, Mitogen, TB1, TB2) 3 (Nil, Mitogen, TB antigen) 3 (Nil, Mitogen, TB antigen) 1

Correction for background IFN-γ level Yes, by subtracting Nil tube IFN-γ value Yes, by subtracting Nil tube IFN-γ value Yes, by subtracting Nil tube IFN-γ value Yes, by subtracting Nil tube IFN-γ value

Positive control Yes, Mitogen tube Yes, Mitogen tube Yes, Mitogen tube Yes, Mitogen tube

ESAT6, CFP-10 ESAT6, CFP-10

ESAT6, CFP-10 ESAT6, CFP-10, TB7.7
Stimulating antigens TB1: long peptides A: ESAT6 peptides
Recombinant proteins Recombinant proteins
TB2: short peptides B: CFP-10 peptides

Antigen stimulation 16–24 hr 20–24 hr 16–24 hr 16–20 hr

IFN-γ detection ELISA ELISA ELISA ELISPOT, single cell resolution

3.9 hr (manual system)

Hands-on time <1 hr <1 hr <1 hr
1.5 hr (automated system)

3 hr for one 96-well plate

plus antigen stimulation 3 hr for one 96-well plate 2 hr for one 96-well plate 5.5 hr plus antigen stimulation
Total processing time
1-2 hr hands on time with automated ELISA plus antigen stimulation plus antigen stimulation plus 4-16 hr for plate drying
systems and results available within 24 hours
Up to 22 tests per 96-well plate
28 tests per 96-well plate
Test capacity or 2 tests per 8-well strip
or 3 tests per 8-well strip 28 tests per 96-well plate 24 tests per 96-well plate
(each patient test requires (8 wells or one strip used for IFN-γ standards)
or 4 tests per 12-well strip (12 wells used for IFN-γ standards) or 2 tests per 8-well strip
3 or 4 wells) Up to 100 samples/run with automated ELISA
(12 wells or one strip used for IFN-γ standards)
systems
Yes, calculation of standard curve and results
Computer required
using QFT-Plus Analysis Software or other Yes, calculation of standard curve and results Yes, calculation of standard curve and results No
to calculate results
software
Result interpretation Positive, negative, indeterminate, invalid Positive, negative, indeterminate, invalid Positive, negative, indeterminate, invalid Positive, negative, borderline, invalid
Yes, required for calculation Yes, required for calculation Yes, required for calculation
Standard curve No
of IFN-γ concentration of IFN-γ concentration of IFN-γ concentration
Type of laboratory Reference laboratory Reference laboratory Reference laboratory Reference laboratory
Operating temperature 2°C to 8°C 2°C to 8°C 2°C to 8°C 2°C to 8°C
Reagent Storage temperature 17°C to 27°C 18°C to 30°C 15°C to 25°C 18°C to 25°C
• 37°C ± 1°C incubator
• 37°C ± 1°C incubator • 37°C ± 1°C incubator • Calibrated pipettes
• Calibrated pipettes
• Calibrated pipettes • Calibrated pipettes • Class II biosafety cabinet (recommended)
• Microplate shaker
• Microplate shaker • Microplate shaker • Centrifuge
• Microplate washer
• Microplate washer • Microplate washer • Equipment to count PBMCs
Required equipment (automated washer recommended)
(automated washer recommended) (automated washer recommended) • Humidified 37 ± 1°C incubator with a 5% CO2
• Microplate reader fitted with 450 nm filter
• Microplate reader • Microplate reader • automatic microtiter plate washer
and 620 nm to 650 nm reference filter
• Centrifuge • Centrifuge • stereomicroscope, magnifying glass
• Optional but recommended:
• Refrigerator • Refrigerator or plate imager
centrifuge for harvesting plasma
Microplate reader, incubator, pipette, Microplate reader requires maintenance Microplate reader requires maintenance Biosafety cabinet requires maintenance
Maintenance and calibration
shaker requires maintenance and calibration and calibration and calibration and certification
Cost Stop TB GDF catalog: $15.90 per test Global access pricing not yet announced Global access pricing not yet announced Stop TB GDF catalog: $10.32-$13.35 per test

48
ANNEXES

50 ANNEX 1:
IGRA IMPLEMENTATION - HIGH LEVEL CHECKLIST
52 ANNEX 2:
QUALITY INDICATORS FOR INTERFERON-GAMMA
RELEASE ASSAYS (IGRAS)
53 ANNEX 3:
IMPACT MEASURES FOR IGRAS

49
 ANNEX 1: IGRA IMPLEMENTATION
HIGH LEVEL CHECKLIST

ANNEX 1

IGRA IMPLEMENTATION - HIGH LEVEL CHECKLIST

→ Policy and planning
• Have roles and responsibilities for coordinating the implementation process been clearly defined?
• Which national guidelines, policies and other materials will need to be updated to include the use of IGRAs
(consider NTP policies and guidelines; screening, diagnostic and preventative treatment algorithms, TB/HIV policies
and guidelines, etc.)?
• Has a stakeholder mapping process been conducted, including all key internal (within government) and external
stakeholders (local and international)?
• What support can partners provide for the implementation process?===
• Has the intended use of IGRAs been decided? Have projections been made for the number of samples to be tested
per year or per site?
• Has a costed implementation plan been developed?
• Have adequate financial resources for capital investments, implementation and projected on-going costs been
secured?

→ Regulatory
• What are the importation requirements for instruments, reagents and supplies for IGRAs?
• What is the regulatory process required for IGRAs?
• Is verification of IGRAs needed for regulatory approval?
• If so, what type of protocol and number of samples are required? Timeline? Where will verification studies
be conducted?
• Is the designated authority (NTP, procurement agency or partners) engaged with manufacturers to support
regulatory processes?

→ Site readiness
• Are adequate laboratory facilities, space and infrastructure available?
• Do facilities, equipment, policies and practices meet biosafety standards?
• Are appropriately trained and competent staff available to conduct IGRAs?
• Which IGRA instruments have been selected and what are the requirements for installation and maintenance?
• Are adequate specimen referral and results reporting systems available?

→ Procurement and supply chain

• Which partners support IGRAs in the country, and what is their scope of activities (how can they contribute
to the transition)?
• Which partners procure instruments and consumables?
• Have manufacturers or distributors been identified who can support implementation, equipment maintenance
(warranties or service contracts) and commodities?
• Is a procurement system available to ensure the availability of reagents and supplies that accounts for procurement
times, consumption rates and shelf-life of reagents?
• What is the planned procurement by MOH and partners for this year?

50
 ANNEX 1: IGRA IMPLEMENTATION
HIGH LEVEL CHECKLIST

IGRA IMPLEMENTATION - HIGH LEVEL CHECKLIST (CONTINUED)

→ Procedures
• Which SOPs and forms will need to be updated or developed?

→ Quality assurance
• Are the essential elements of a quality assurance system in place at the testing site?
• Are protocols in place to conduct and document quality checks of each step of the IGRA process and ensure the use
of positive and negative controls?
• Is an external quality assessment programme in place?
• Which partners can assist with proficiency testing, supervisory visits and re-checking of samples?
• Have quality (performance) indicators been defined and appropriate data collection tools developed?

→ Recording and reporting

• Are revisions of the current data collection form and request for laboratory testing form required?
• Is revision of forms for reporting the results of laboratory testing needed?
• Is a revision of laboratory, clinical or surveillance registers needed?
• If an electronic laboratory information system is in use, what updates will be required?
• If an electronic recording and reporting system is in place, what updates will be required?

→ Training
• Have terms of reference and competency-based job descriptions been developed for key staff?
• Is a national approved training curriculum available?
• Who is responsible and what is the process for updating training materials for laboratory, clinical and programme
staff?
• Is the approved curriculum used for all trainings, including those delivered by partners?
• Are standard procedures used to assess and document the competence of all staff involved in IGRAs?

→ Monitoring the transition

• What changes to M&E tools and processes would be required to enable monitoring of additional indicators
(i.e. progress indicators and laboratory indicators)?
• What support can partners provide in monitoring of new algorithms and adherence to guidelines at sites?

51
 ANNEX 2: QUALITY INDICATORS
FOR INTERFERON-GAMMA RELEASE ASSAYS
(IGRAS)

ANNEX 2

QUALITY INDICATORS FOR INTERFERON-GAMMA RELEASE ASSAYS (IGRAS)

Targets should be set for all indicators that are monitored, and any unexplained change in quality indicators,
such as increase in error rates or a change in positivity rate should be documented and investigated. Targets may
need to be adjusted as information on the use of these tests in routine diagnostic settings become available.

INDICATOR DESCRIPTION TARGET

Number of samples received per month

Number and proportion of tests

performed per month

Number and proportion of rejected Number of rejected samples / Total number

<5%
samplesA of specimens received

Number and proportion of samples Number of samples with positive results / Dependent
with positive results Number of samples tested on population tested

Number and proportion of samples Number of samples with borderline results / Dependent
with borderline results (T-Spot.TB only) Number of samples tested on population tested

Number and proportion of test runs

Number of test runs with valid standard curves /
with valid IFN-γ standard curves >95%
Number of test runs
(ELISA-based tests)

Number and proportion of samples Number of samples with indeterminate results /

<5%
with indeterminate results Number of samples tested

Number and proportion of samples Number of specimens with errors/ Total number
<3% (Xpert)
with errorsB of samples tested

Number and proportion of samples Number of samples with invalid results / Total
<1% (Xpert)
with invalid results number of samples tested

Number and proportion of samples tested

Number and proportion of samples tested
with an IGRA for which a result was
with an IGRA for which a result was reported
reported within the target turnaround time >90%
within the target turnaround time / Total
(i.e. time from blood collection to reporting
number of samples tested
of results)C

A Stratify by reason rejected (insufficient volume, hemolyzed, incorrect collection tube, received past cutoff) to enable troubleshooting.
B Errors should be stratified by type to enable troubleshooting.
C Target turnaround time should be set by the program taking into consideration sample transport, testing schedules and test characteristics.
For troubleshooting, analyse time from blood collection to receipt in the laboratory, time from blood collection to initiation of the immune stimulation,
and time for the IFN-γ detection step (e.g., ELISA or ELISPOT).

52
 ANNEX 3: IMPACT MEASURES FOR IGRAS

ANNEX 3

IMPACT MEASURES FOR IGRAS

For each of the below indicators, stratification by target population and risk of exposure (e.g. household contacts
and children under 5 years) or risk of progression (e.g. PLHIV) will allow for optimal assessment of the impact
of activities:
• Number and proportion of eligible persons that are tested for TB infection using an IGRA
• Number and proportion of tested persons with a positive IGRA result
• Number and proportion of tested persons with a positive IGRA result placed on TPT
(proportion = number of tested persons with a positive IGRA placed on TPT) / number of persons with a positive
IGRA)
• Number and proportion of persons placed on TPT that had a positive IGRA test
(proportion = number of persons placed on TPT that had a positive IGRA result / number of persons placed on TPT)

53
 SUGGESTED READING

1. WHO consolidated guidelines on tuberculosis.

Module 3. Diagnosis tests for TB infection.
Geneva: World Health Organization, 2022.
https://www.who.int/publications/i/item/9789240056084

2. WHO operational handbook on tuberculosis.

Module 3. Diagnosis tests for TB infection.
Geneva: World Health Organization, 2022.
https://www.who.int/publications/i/item/9789240058347

3. WHO operational handbook on tuberculosis.

Module 3: diagnosis. Rapid diagnostics for tuberculosis detection.
Geneva: World Health Organization, 2021.
https://www.who.int/publications/i/item/who-operational-handbook-on-
tuberculosis-module-3-diagnosis---rapid-diagnostics-for-tuberculosis-
detection

4. Framework for the evaluation of new tests for tuberculosis infection.

Geneva: World Health Organization, 2020.
https://apps.who.int/iris/rest/bitstreams/1288536/retrieve

5. Use of alternative interferon-gamma release assays for the diagnosis

of TB infection: WHO policy statement.
Geneva: World Health Organization, 2022.
https://apps.who.int/iris/bitstream/handle/10665/351191/
9789240042346-eng.pdf

6. Alsdurf H, Hill PC, Matteelli A, Getahun H, Menzies D.

The cascade of care in diagnosis and treatment of latent tuberculosis
infection: a systematic review and meta-analysis.
Lancet Infect Dis. 2016;16(11):1269–78.

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