World Journal of AIDS, 2012, 2, 222-225 Published Online September 2012 (
External Quality Assurance Scheme in a National
Reference Laboratory for HIV Testing in South India
Kamala Mary Sushi, Thatchinamoorthy Gopal, Saramma Mini Jacob*, Ganesan Arumugam,
Anitha Durairaj
Department of Experimental Medicine, TN Dr. MGR Medical University, Chennai, India.
Email: *
Received May 17th, 2012; revised July 10th, 2012; accepted July 18th, 2012
Background: The Department of Experimental Medicine functions as the National Reference Laboratory (NRL) for
HIV testing covering 11 Medical Colleges (State Reference Laboratories SRL) & 723 subcenters i.e. Integrated Coun-
seling & Testing Centres and Blood Banks. The External Quality Assurance Scheme (EQAS) in NRL implements
Quality Control (QC) Testing, Proficiency panel testing and training programs. Materials & Method: 9419 samples
(4393 HIV negative/5026 HIV positive) were tested for QC. All the samples were tested using HIV rapid test (Com-
bAids) and HIV positives alone were tested using Tridot and EIA Comb. The QC samples consisted of 20% negative
and all positives. All the 723 subcenters were provided with 5 coded plasma samples (3 reactive & 2 negative) for pro-
ficiency testing using rapid tests. The aliquot panel (500 μl) were provided twice a year for testing to monitor the labo-
ratory performance. Results: Out of 9419 samples tested for QC, 9371 (99.49%) reported correct results and 48 (0.50%)
discordant results. Out of 48 samples 26 (0.27%) were false positives and 22 (0.23%) false negative. Mislabeling, sam-
ple contamination, leaking vials, transcriptional errors, tests that were not performed correctly were identified. For pro-
ficiency testing 91.8% reported test results. 645 (97.13%) reported correct results & 19 (2.86%) incorrect results. Out of
19 samples 7 (1.05%) were false positive & 12 (1.80%) were false negative. Hands on training were provided and the
19 discordant centers reported correct results on retesting. Conclusion: Significant progress in establishing a well coor-
dinated HIV Laboratory network of NRL and SRLs had been developed. However the HIV testing and Quality Assur-
ance needs to be strengthened towards certification.
Keywords: Quality Assurance; HIV Testing; South India
1. Introduction
Quality Assurance is the series of procedures that ensure
that a correct result is achieved in a standard, reproduci-
ble and traceable manner [1]. It is achieved by a series of
processes that assure the most accurate and highest qual-
ity result. To achieve a high quality result input from all
members of every laboratory in a testing network is re-
quired. All laboratory personnel should be aware of the
necessity for quality performance. This requires con-
tinuous education throughout the testing system.
The Department of Experimental Medicine of the TN
Dr. MGR Medical University functions as one of the
National Reference Laboratory (NRL) in the External
Quality Control Scheme (EQAS) under the National
AIDS Control Organization (NACO), Government of
India for HIV testing. The NRL is in charge of eleven
State Reference Laboratories (SRL) and 723 subcenters
i.e., Intergrated Counselling and Testing Centers (ICTC)
and Blood Banks (BB).
The NRL tests Quality Control (QC) samples i.e.,
every 20th HIV negative and all positives. Apart from
QC testing, preparation of panels, hands on training and
training programs on EQAS and QC testing for sentinel
surveillance are the other activities.
The Diagnostic tests to detect antibodies to HIV have
sensitivity and specificity which are not absolute. In all
these tests we have false negative and false positive re-
sults which are inherent and cannot be avoided. Thus the
validity of diagnostic test results is dependent to a very
large extent on the quality of the technical conditions
under which the tests are performed. Meaning thereby,
consistent reproduction of reliable results requires a
stringent overall Quality Assurance program which would
control technical conditions before, during and after each
The Center for Disease control (CDC) developed
guidelines for implementing and operating QA programs.
*Corresponding author.
Copyright © 2012 SciRes. WJA
External Quality Assurance Scheme in a National Reference Laboratory for HIV Testing in South India 223
It recommends testing sites participate in external quality
assessments ie Proficiency testing of panels, competency
assessments and QA monitoring by outside organization
observing the testing [2]. The National Reference Labo-
ratory (NRL) implements the External Quality Assurance
Program for HIV testing to provide standard quality re-
2. Material & Methods
This is a retrospective study. The Quality Control (QC)
samples were received from the 11 State Reference
Laboratories (SRL) by courier service and hand delivery
from 2007 to 2008. The SRLs test the samples in their
respective centers. After testing the QC samples alone
are sent to the NRL. Every 20th HIV negative sample and
all HIV positive samples from the State Reference Labo-
ratories are tested as QC samples in the NRL. 9419 sam-
ples were received in the NRL for QC testing (5026 posi-
tive and 4393 negative). In QC testing all the samples are
retested using CombAids (Span Diagnostics Ltd, Surat,
India) as the 1st rapid kit. It employs the same principle
as EIA thereby the immobilized antigen antibody com-
plex is visualized by means of colour producing (chro-
mogenic reaction). Each comb was observed for the con-
trol and the test results. The HIV positives alone were
retested by the 2nd kit HIV Tridot (J. Mitra & Co, India)
and the 3rd rapid EIA comb (J. Mitra & Co). NACO
strategy of using 3 rapids for HIV testing was followed
[3]. Insufficient and hemolysed samples, samples that
were not properly labeled were excluded and informed to
the testing centers. Apart from the routine samples and
kit controls Internal Quality Control samples (known
HIV positive & negative) were used during each run. The
test kits were provided by the APSACS (Andhra Pradesh
State AIDS Control Society). All discordant samples
were discussed with the testing center and were followed
up with hands on training for the laboratory technicians.
It is a continuous process where the NRL plays a key role
in monitoring and evaluating the SRLs.
Apart from QC testing two training programs were
conducted twice a year for the SRLs. One Microbiologist
and one laboratory technician represented each SRL. The
training comprises lectures, hands on training in HIV
testing and panel distribution. The SRL’s & 723 subcen-
tres were provided with 5 panels (3 HIV positives & 2
negative coded samples) during the training program.
Panels were prepared by NRL procuring blood bags or
plasma from blood banks. The samples were character-
ized using ELISA and HIV rapid tests. The panels were
validated by sending them to the Apex Laboratory before
the training program. A report was sent from the Apex
Lab to NRL informing the results and samples that had
concordant results were used for proficiency panel test-
ing. Aliquots were further prepared and stored in –80˚C.
The SRL’s in turn conducted a one day training program
for their sub centers and provided the panels. The reports
of the 723 subcenters provided a feedback and this en-
abled the NRL to train them periodically and improve
their performance in HIV testing.
3. Results
Nine thousand four hundred and nineteen Quality control
(QC) samples were tested at the National Reference
Laboratory during a period of 2 years. Out of 9419, 5026
samples were HIV positives (53.3%) and 4393 were HIV
negative samples. The samples were retested using three
different HIV rapids test kits. The 9419 samples were
tested using Comb AIDS kit as the first rapid kit.
Out of 9419 samples tested, 9371 reported concordant
results. 4367 QC samples were HIV negative and 5004
QC were positive for HIV. 48 reported discordant results.
26 samples were false positive and 22 were false nega-
tives (Table 1). Out of 5026 QC positive samples re-
ported from the SRL, 22 samples were false negative and
5004 were HIV positive. Similarly out of 4393 QC nega-
tive samples 26 tested false positive and the remaining
4367 were negative (Table 2).
The QC positive samples, 26 false positive and 22
false negative samples were retested using the second
HIV rapid test Tridot. 5004 samples were positive. 26
reported false positive and 22 false negative in Tridot.
The above QC samples were again retested with the third
HIV rapid test kit and similar results were observed. 48
discordant samples reported the same results in all the
three rapid kits. The QC sample result & discordant sam-
ples were reported to the respective centers. The main
reasons for discordant samples were contamination of
samples, pipette tip contamination, labeling error, leaking
vials and transcriptional errors.
Table 1. Concordant and discordant samples tested for
Quality Control.
Total no of QC
samples Discordant samples
9419 9371 (99.4%)48 (0.50%)
False positive False negative
26 22
Table 2. Samples tested at SRL and NRL.
No. of samplesCombAids TRIDOT EIAComb
439326 436726 26
5026 5004 22 5004 22 5004 22
Copyright © 2012 SciRes. WJA
External Quality Assurance Scheme in a National Reference Laboratory for HIV Testing in South India
Twice a year proficiency testing panels (coded sam-
ples) were distributed to SRL’s during the training pro-
gram. Out of 723 subcenters, 664 (91.8%) reported the
panel results to the NRL. 59 (8.16%) centers did not send
the report. Out of 664 subcentres, 645 (97.1%) reported
correct results and 19 (2.86%) reported incorrect results.
Out of 19 samples 7 (1.05%) reported false positive & 12
(1.80%) false negative. Contamination of samples was
the major reason for incorrect results. Hands on training
on HIV testing were provided to the laboratory techni-
cians in 19 centers as follow up. New set of plasma pan-
els (5 Nos) were provided. All the 19 centres reported
correct results.
4. Discussion
The NRL is involved in Quality Control testing, profi-
ciency panels, conducting training programs and provid-
ing hands on training for HIV testing. It is also involved
in sentinel surveillance QC testing which is conducted
annually by NACO. Quality Assurance program ensures
high level of performance of HIV assays [4]. As a NRL
the Quality Assurance program ensures the participating
centers are correctly testing, getting consistent results
and it checks their validity of results.
In India the EQAS program had been implemented by
NACO since 2000. It functions with 1 Apex Laboratory
and 13 National Reference Laboratories and thousands of
sub centers covering the entire Government Medical
Colleges, Hospitals, blood banks and primary health
centers in each state. This EQAS program is linked with
the respective State AIDS Control Society (SACS) in
each state for the effective functioning under the NACO.
Out of 9419 samples tested for QC, 48 samples had
discordant results: 26 false positive and 22 false negative.
In the follow up training program, they were instructed to
follow the Standard Operating Procedures (SOP), use of
correct pipetting technique, use of single tip for each
sample, avoid transcriptional errors and prevent sample
contamination. Participation in proficiency testing is a
key component of any Laboratory Quality assurance
program, whether available locally, nationally or interna-
tionally [5].
Quality Assurance program should be in place to con-
tinuously assess and improve the performance of Labo-
ratory results. It is important for the physicians in guid-
ing the patient for treatment and for further management.
All aspects of sample handling right from the arrival to
reporting must be monitored, documented and subjected
to quality control procedures. It is also suggested that
regular audits of laboratory procedures and reviewing
incident reports must be carried out by the senior staff
who are in charge and should be discussed with the Di-
rector or the Head of the Laboratory [5,6].
The Standard Operating Procedures (SOP) is an im-
portant component of the Quality Assurance program.
All the members of the laboratory should be familiar
with the procedures. The SOP’s are reviewed every year
and no deviations from the procedures in order to get
correct results.
Reagent and equipment performance must be moni-
tored over time to detect any changes in quality and in-
tegrity [7]. The storage of reagents and expiry date too
plays an important role in the performance of testing. If
controls are not working faint spots or lines may develop.
It will lead to misinterpretation of results. Calibrating
pipettes and centrifuges annually should be done in the
laboratory. The temperatures of the refrigerators are
monitored by a digital thermometer where HIV testing
kits are stored. Expired kits should not be used.
In Catalonia, Spain a survey on HIV testing was un-
dertaken to assess the quality of HIV testing. HIV
specimens were identified by specific labels, extracting
new specimens for a second test to confirm, or failing to
guarantee the confidentiality of results was found [8]. In
a QA program the pretesting, testing and post testing
should be monitored, observed and evaluated. The rec-
ommendations will play an important role in improving
the centers.
In China the National AIDS Reference Laboratory
(NARL) provides quality assurance through technical,
bio safety and managerial trainings, periodic proficiency
testing, on site supervisory inspections and commercial
serological kit evaluations [9]. The 9419 samples tested
for QC provided a feedback mechanism on the perform-
ance of the respective centers. It provided an opportunity
to pick up discordant samples or it could have been left
undetected. After the implementation of the EQAS pro-
gram the standards of HIV testing had been improved.
Rapid test with immediate test result are popular in the
US and Canada. It requires good laboratory quality con-
trol practices. The laboratory must be backed by a license
to provide confirmation of positive results and resolution
for indeterminate results. Quality Assurance and partici-
pation in HIV proficiency testing ensures the accurate,
timely and clinically relevant laboratory results [7]
The EQAS training twice a year and proficiency test-
ing was aimed to monitor testing at SRL’s who in turn
apply the same methods to the subcenters. Out of 723
subcenters provided with proficiency panel, 664 (91.8%)
reported the results. 8.16% did not report. The main rea-
son being the center did not participate in the training
program. 97.1% reported correct results. 19 (2.86%) re-
ported incorrect results. Hands on training were provided
to 19 centers and they were provided with new set of
proficiency panels. All the 19 centers reported correct
In Poland there is no guidelines related to HIV diag-
nosis quality assurance and control. Hence developing of
Copyright © 2012 SciRes. WJA
External Quality Assurance Scheme in a National Reference Laboratory for HIV Testing in South India
Copyright © 2012 SciRes. WJA
a National Unified quality control system based with a
central institution is highly desirable. This was based on
the survey conducted to assess the laboratory perform-
ance. It was also suggested certification be made manda-
tory for all diagnostic laboratories [10]. The aim was to
improve the reliability of results among the clinicians
and the patients.
The proficiency testing provided an opportunity for the
subcenters to know their abilities and improve their per-
formance. They were also provided with forms to fill up
the results ie, kit name, lot No, expiry dates, results of
controls and samples and the final interpretation of re-
sults. These forms were provided by the Apex Labora-
tory and it provided an opportunity for the Lab personnel
to read and fill in the forms. All the 723 subcenters were
provided with unique codes identifying with the SRL,
District and their VCTC, BB centers.
In India five laboratories were identified as Regional
Institutes (RIs) to monitor and supervise the Quality con-
trol practices and assurances in the sentinel surveillance
testing covering five states. The testing laboratories were
adhering to the standard Operating Procedures. Concor-
dance of test result between the RI and laboratories was
high. The lacunae and the recommendations were put
forward for the future surveillance [11].
5. Conclusions
Significant progress had been made in establishing a well
coordinated HIV Laboratory network through EQAS. In
this program it ensures mistakes may be avoided by
proper sample handling, labeling, testing and reporting.
The consistency of performance is maintained. The Qual-
ity Assurance system with proficiency testing, quality
control testing, training programs and on-site monitoring
strengthens the participating laboratories and provides
opportunity for improvement.
NABL certification is in process in the EQAS program.
The certification process which covers the entire labora-
tory performance will provide opportunities for filling
the gaps and strengthening their performance.
6. Acknowledgements
The authors are thankful to the National AIDS Control
Organization and Andhra Pradesh State AIDS Control
Society for supplying HIV tests kits.
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