External Quality Assurance Scheme in a National Reference Laboratory for HIV Testing in South India

doi:10.4236/wja.2012.23028

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World Journal of AIDS, 2012, 2, 222-225

http://dx.doi.org/10.4236/wja.2012.23028 Published Online September 2012 (http://www.SciRP.org/journal/wja)

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: *[email protected]

Received May 17th, 2012; revised July 10th, 2012; accepted July 18th, 2012

ABSTRACT

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

assay.

The Center for Disease control (CDC) developed

guidelines for implementing and operating QA programs.

*Corresponding author.

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-

sults.

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

Concordant

samples Discordant samples

9419 9371 (99.4%)48 (0.50%)

False positive False negative

26 22

Table 2. Samples tested at SRL and NRL.

SRL NRL

No. of samplesCombAids TRIDOT EIAComb

HIV+ HIV−HIV+ HIV−HIV+ HIV− HIV+ HIV−

439326 436726 26

5026 5004 22 5004 22 5004 22

External Quality Assurance Scheme in a National Reference Laboratory for HIV Testing in South India

224

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

results.

In Poland there is no guidelines related to HIV diag-

nosis quality assurance and control. Hence developing of

External Quality Assurance Scheme in a National Reference Laboratory for HIV Testing in South India

225

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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