External quality assessment program on CD4+ T-lymphocyte counts for persons with HIV/AIDS in Thailand: history and accomplishments.

Abstract

A CD4 count External Quality Assessment (EQA) program is important for the clinical monitoring of persons infected with HIV/AIDS. The purpose of the present study was to evaluate the CD4 EQA performance program of the flow cytometer laboratories that perform routine CD4 counts for these patients in Thailand. Stabilized whole blood samples were sent to participating laboratories to determine the percentage and absolute counts of CD4+ T-lymphocytes using their routine procedures. The data were analyzed and reports sent to the participants within one month. Most participating laboratories produced results that were within two standard deviations (SD) of the mean, while the average inter-laboratory coefficients of variation were less than 8% for CD4+ T-lymphocytes. This program was found to improve the reliability of CD4+ T-lymphocyte determinations. This test is becoming increasingly important as Thailand and other Southeast Asian countries scale up their national programs that provide access to antiretroviral therapy for persons living with HIV/AIDS.

Full text

Page 1

ASIAN PACIFIC JOURNAL OF ALLERGY AND IMMUNOLOGY (2009) 27: 225-232


From the 1Center of Excellence for Flow Cytometry, Office for
Research and Development, Faculty of Medicine Siriraj Hospital,
Mahidol University, Bangkok, Thailand, 2Faculty of Public Health,
Mahidol University, Bangkok, 3South East Asia Research Colla-
boration with Hawaii, Bangkok, Thailand.
Correspondence: Kovit Pattanapanyasat
E-mail: grkpy@mahidol.ac.th
SUMMARY A CD4 count External Quality Assessment (EQA) program is important for the clinical monitoring of
persons infected with HIV/AIDS. The purpose of the present study was to evaluate the CD4 EQA performance
program of the flow cytometer laboratories that perform routine CD4 counts for these patients in Thailand. Stabi-
lized whole blood samples were sent to participating laboratories to determine the percentage and absolute counts
of CD4+ T-lymphocytes using their routine procedures. The data were analyzed and reports sent to the participants
within one month. Most participating laboratories produced results that were within two standard deviations (SD) of
the mean, while the average inter-laboratory coefficients of variation were less than 8% for CD4+ T-lymphocytes.
This program was found to improve the reliability of CD4+ T-lymphocyte determinations. This test is becoming in-
creasingly important as Thailand and other Southeast Asian countries scale up their national programs that provide
access to antiretroviral therapy for persons living with HIV/AIDS.


External Quality Assessment Program
on CD4+ T-Lymphocyte Counts for Per-
sons with HIV/AIDS in Thailand: History
and Accomplishments


Vallerut Pobkeeree2, Surada Lerdwana1, Umaporn Siangphoe3, Egarit Noulsri1, Korakot Polsrila1,
Somboon Nookhai1 and Kovit Pattanapanyasat1*





The Thailand National AIDS Program
(NAP) is responsible for at least 100,000 HIV-
infected people among the national population of 65
million. New annual HIV infections decreased from
17,000 in 2004 to around 14,000 in 2007.1 However,
total HIV infections are currently at least 700,000.2

CD4+ T-lymphocytes are a primary target of
HIV and are preferentially depleted throughout the
course of the disease.3 Thus, they are markers for
both the prognosis and therapeutic monitoring of the
disease. Whole blood CD4+T-lymphocyte levels are
measured using flow cytometric immunopheno-
typing. This is widely accepted as the standard meth-
od for this purpose due to its superior accuracy, pre-
cision and reproducibility.4

The use or misuse of CD4+T-lymphocyte
measurements has a crucial impact on the effective
management of HIV/AIDS patients. It is essential
that accurate daily Internal Quality Control (IQC)
and proficiency testing or External Quality Assess-

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226 POBKEEREE, ET AL.

226
ment (EQA) programs are employed to ensure relia-
ble results. Satisfactory performances in CD4+T-
lymphocyte EQA are recommended for HIV re-
search and clinical trial programs within many parts
of the world, including Thailand.

The limited budget for the Antiretroviral
Therapy (ART) Program has hampered the EQA
program in Thailand. Previously, the Thai govern-
ment requested funding from the Global Fund to
Fight AIDS, TB and Malaria for ART care and
treatment, but this funding was not sufficient to cov-
er all cases of the disease. Consequently, many in-
fected people did not have access to the NAP to fol-
low up their CD4+T-lymphocyte measurements.
This testing is an important monitoring tool used in
HIV treatment in Thailand. An alternative mea-
surement, viral load testing, is far more expensive.
Newly-infected patients also should be introduced to
NAP and tested for their CD4 baseline values, so that
these and other infected persons receive ART and
follow-up CD4+ T-lymphocyte count determinations.

CD4+ T-lymphocyte counts are performed
by flow cytometry laboratories located in public
health hospitals throughout the country. Sixteen flow
cytometers were purchased by the Ministry of Public
Health in 1991, and there are currently over 150 such
machines performing routine CD4+T-lymphocyte
count tests. All regional, provincial and some large
district hospitals provide testing services for nearby
hospitals that lack this equipment. More than half the
flow cytometers in the country can perform the dual
platform (DP) technique, although the single plat-
form (SP) technique is also commonly employed.5
SP is a simplified flow cytometer which utilizes a
single instrument.3,5 It provides an absolute CD4
values with less variation than the DP method. The
DP technique uses two instruments: a flow cytometer
for determining the percent CD4+T-lymphocytes
among all lymphocytes, and a hematologic cell ana-
lyzer which determines absolute lymphocyte num-
bers, and hence the measurements show more varia-
tion than the SP approach. The only SP flow cytome-
ter used in the country is the FACSCount. The origi-
nal 16 flow cytometers were bought from Beckton
Dickinson Biosciences (BDB, San Jose, CA, USA)
for the DP technique. Later, the Ministry received
additional funding and subsequently purchased both
DP and SP machines. Thailand presently has approx-
imately 150 flow cytometers. The DP machines ac-
count for 100 of these while the remaining 50 use the
SP technique (FACSCount).

The Center of Excellence for Flow Cytome-
try is the sole CD4 EQA provider in Thailand and
participate in two international CD4 EQA programs:
Quality Assessment and Standardization for Immo-
nulogical Measures Relevant to HIV/AIDS (QASI,
Canada-based program) and United Kingdom Na-
tional Quality Assessment Services for Lymphocyte
Immunophenotyping (UKNEQAS).6,7 The QASI
panel is shipped three times a year and free of charge,
while the UKNEQAS annual fee and shipment
charges are relatively expensive. UKNEQAS panels
are delivered six times a year. For the past few years,
the quality of the UKNEQAS samples was substan-
dard, possibly as a result of temperature differences
between the two countries. These samples necessi-
tated the use of the lyse-and-wash procedure not
usually performed routinely.

The CD4 EQA program has two main objec-
tives. Firstly, it must conduct a low-cost national
EQA and standardization of flow cytometer CD4+T-
lymphocyte determinations in HIV/AIDS patients.
Secondly, the EQA provider should assist in the
country’s flow cytometer laboratory performance
evaluations, and incorporate scientifically and educa-
tionally-based schemes in order to monitor their im-
provement. Towards the middle of 2008, there were
30 EQA trials with 130 participating laboratories
mostly from Ministry of Public Health. In this report,
we summarize and discuss the history and the find-
ings from the last 6 years of our EQA program on
CD4+ T-lymphocyte counts.

MATERIALS AND METHODS

Aliquots of the same batch of stabilized
EQA whole blood were distributed to participating
laboratories throughout the country. The EQA blood
sample panels were prepared from healthy donors’
blood and stabilized according to a fixative method
developed at the Center of Excellence for Flow
Cytometry. Each batch of EQA blood was evaluated
to ensure that immunophenotypic characteristics
have been maintained during handling and transpor-
tation. Laboratories were instructed to acquire and
analyze the EQA blood samples using their standard

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227

227
operating procedure, which are normally followed
the manufacturer’s instructions.

A shipping schedule is sent annually. Six
whole-blood trials are conducted per year. The first
CD4 EQA trial is labeled Trial 01. Each trial consists
of different panels based according to the number of
participating members. As of mid-2008, the program
had reached Trial 30. The panels were sent by post at
ambient temperature within 1-3 days and then stored
at 2-8C until testing require. Four weeks were per-
mitted for the laboratories to conduct tests and to re-
turn the results to the program provider.

After the participating laboratories received
an EQA tube along with brief instructions and a
blank report form, they recorded the date received
and the integrity of the sample. Flow cytometer la-
boratories performed the CD4+ T-lymphocyte test-
ing either by a lyse-and-wash procedure, whereas
other laboratories used a lyse-no-wash procedure.
The samples were routinely stained with three-color
and two-color monoclonal antibodies for the DP and
SP techniques respectively. Laboratories using SP
technique were asked to report the absolute CD4+T-
lymphocyte count while laboratories using DP tech-
nique were required to obtain a complete blood count
using a hematology analyzer followed by %CD4+T-
lymphocyte measurement. Both the percentage and
absolute counts of CD4+T-lymphocytes were often
determined by DP users. Most flow cytometer labor-
atories use automatic lymphocyte gating and appro-
priate BDB or other software to analyze the data.
Submitted results not only included CD4+T-
lymphocytes, but also CD3+ pan T-lymphocytes and
CD8+ suppressor T-lymphocytes were also accepted
and analyzed but only CD4+ T-lymphocytes are re-
ported here.

The statistical analysis of the most important
parameter (CD4+T-lymphocytes) was analyzed using
Microsoft Excel (Microsoft Corp., Redmond, WA).
P-values (< 0.05) were calculated for the response
rates of both platforms to show the statistical signi-
ficance of differences by chi-square. The mean and
standard deviation (SD) were used to group the
ranges of CD4+T-lymphocyte values and percentage
of coefficient of variation (%CV) for both DP and
SP techniques. Laboratories with results > 2SD from
the pool mean results were considered as outliers and
were removed. The trimmed data were reanalyzed to
obtain a trimmed mean and a SD for CD4+T-
lymphocyte values. Each laboratory result was com-
pared directly to the trimmed SD value. Data of
mean ± SD for each individual trial were depicted
using the Levy-Jennings plot. Performance assess-
ment expressed by the average %CV from Trails 12
to 30 for all participating SP laboratories and Trials
1 to 30 for all participating DP laboratories were also
demonstrated by longitudinal plots.

RESULTS

Thirty CD4 EQA trials were delivered to
participating laboratories between 2002 and 2008.
The results were returned on a monthly basis as re-
quired by the accompanying leaflet for each panel.
Data was collected and analyzed using spreadsheet
software.

Since SP protocols became available only 3
years ago, DP data was acquired exclusively for the
initial Trials 01 to 11. The overall response rates for
both platforms were high throughout the course of
the trials (2002-2008). During the first year of SP
availability, there was no significant difference in the
response rates for each platform. However, in the
next three years there were significant differences, as
SP had a low response rate of 84%. This may be be-
cause some laboratories with access to both SP and
DP cytometry did not use both methods due to time
and budget constraints. Also, they may not use both
platforms routinely, or in some cases only one ma-
chine was accessed as a backup in busy hospital la-
boratories or medical institutes. Furthermore, the DP
flow cytometers were originally bought and distri-
buted initially, whereas SP flow cytometers were
purchased at a later time.

A total of 2,307 samples were sent to SP and
DP users. The absolute and %CD4+T-lymphocytes
values were both within the mean ± 2SD for 2,209 of
these samples. Table 1 indicates significant differ-
ences between the CD4 EQA value distributions for
both platforms. However, more than 70% of these
were within the mean ± 1SD.

The average coefficient of variation (%CV)
of SP for 19 trials (Trials 12 to 30) was 5.6%.
The %CV was consistent (Fig. 1A) but two individu-

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228 POBKEEREE, ET AL.

228

Table 1 CD4 EQA value distribution and platform technique

Results Both platforms Single platform Dual platform
No. samples
No. trials
No. sites
2,307
30
130
673 (29.2%)
19 (63.3%)
49 (55.7%)
468.2 ± 194.1
(absolute CD4 cells/mm3)

561 (83.4)
52 (7.7)
60 (8.9)
< 0.001
1,634 (70.8%)
30 (100.0%)
81 (92.1%)
35.9 ± 10.1
(% CD4)

1,095 (67.0)
501 (30.7)
38 (2.3)
< 0.001
Mean values ± SD
-
No. samples within SD groups
a) Within ± 1SD
b) Between ± 1SD and ± 2SD
c) Outside ± 2SD
P-value: a) vs. b) and c)

1,656 (71.8)
553 (24.0)
98 (4.2)
< 0.001

al trials showed high %CV, those being Trial 20
(12.8%) and Trial 22 (11.6%). Experimental pipet-
ting errors during sample preparation for FACS-
Count8 may account for this. For example, laboratory
personnel may not have conducted tests using BDB-
provided electronic pipettes. In addition, machines
may not have been correctly calibrated. The results
for the DP technique were inconsistent, showing a
high %CV in the first four trials (Fig. 1B). The av-
erage %CV was 18.9%, but dropped to < 10 %CV
after trail 5.

The results were analyzed and depicted us-
ing a Levy-Jennings plot. Fig. 2 illustrates EQA Trial
07 (% CD4+T-lymphocytes), which was conducted
in 2003. Any outliers over ±2 SD from the mean
were deemed unacceptable. Participants were coded
on the X axis and new members were added each
year, as shown on the X axis. The right hand scale
shows the consensus mean of %CD4+T-lymphocytes
±1 SD and ±2 SD. The left hand scale shows
the %CD4+T-lymphocytes. Fig. 3 illustrates EQA
Trial 15, conducted in 2005, which indicates that la-
boratories participating for longer durations within
the EQA program show an increased performance in
these later trials.

DISCUSSION

We separated the analysis into two platforms,
single and dual. In the first 11 trials, the %CD4+T-
lymphocytes were analyzed since the DP method
was used exclusively. Later, both DP and SP proto-
cols were used, producing both %CD4+T-
lymphocyte and absolute CD4+T-lymphocyte data.
The % value was required rather than absolute value
analysis only, because up to two thirds of flow cyto-
meter users performed the DP technique, thus auto-
matically providing % CD4+T-lymphocyte values.

The SP technique yields more accurate abso-
lute CD4 values than the DP technique.8,11 This is
due to the variations that occur with the use of hema-
tology analyzers during the DP process.6 However,
following the development of new BDB reagents and
software12, BDB intends to upgrade all FACSCount
software in Thailand so that both % CD4 values and
absolute values can be acquired and printed concur-
rently. At present, however, Thailand will continue
to use the DP technique as its conventional technolo-
gy, unlike other countries such as Brazil or various
African countries.13,14 The DP protocol was the first
one to be used in Thailand, and laboratory personnel
are familiar in using its protocol. Moreover, approx-
imately half the DP machines in the country contain
auto-loading for high throughput laboratories. Al-
though the SP method results in less inter-laboratory
variability, the higher response rate of DP and de-
clining response rate of SP (data not shown) is sup-
portive of our prediction that DP will be preferable
to SP. It should be noted that in the absence of DP
technology, SP laboratories should maintain a rigor-
ous testing of EQA panels when they received.

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229

Fig. 1 Longitudinal performance assessment of the coefficient of variation (%CV) of SP technique
obtained from Trials 12 to 30 (A); %CV of DP technique obtained from Trials 1 to 30 (B).
The %CV measurements for the SP tech-
nique in Trials 12-30 were low and quite consistent
(Fig. 1A). However, two trials (Trials 20 and 22)
yielded a high %CV, possible due to the aforemen-
tioned equipment error or because the means of the
absolute CD4+T-lymphocyte values were the least
(less than 300 cells/mm3) among the trials. This is in
accordance with previous studies7,15,16 showing a
high %CV when CD4+T-lymphocyte measurements
were at low or medium levels. As mentioned pre-
viously, one third of the flow cytometers used in the
country are SP machines (FACSCount), which con-
tain one forward scatter feature along with PE and
PE-Cy5 fluorochromes. The machines also do not
serve high throughput laboratories and therefore tend
to be used in small- and medium-sized laboratories.
Laboratory personnel, who are medical technologists,
usually conduct CD4+T-lymphocyte count testing
and have more experience with the DP technique.
They typically perform the test at specific times dur-
ing the week (e.g. once or twice a week, or every
second day) rather than daily due to other time and
work commitments. The highest number of tests per-
formed is approximately 1,000 per month at regional,

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230 POBKEEREE, ET AL.

230
48
50
52
54
56
58
60
62
64
13579
11 1315 1719 21
Site Number
2325 27293133 353739 41434547
%CD4+ T-Lymphocytes
Mean = 55.73
-1SD
-2SD
+1SD
+2SD
Figure 2.



Fig. 2 Levy-Jennings plot demonstrating % CD4+ T-lymphocytes obtained from Trial 7 in 2003.
18
23
28
33
38
43
48
159
131721 252933 37 41 454953 5761
Site Number
%CD4+ T-Lymphocytes
Mean = 32.65
-1SD
-2SD
+1SD
+2SD
Figure 3.


Fig. 3 Levy-Jennings plot demonstrating % CD4+T-lymphocytes obtained from Trial 15 in 2005.
provincial or medical institute laboratories, far less
than in African regional laboratories.13

Results in Fig. 1B show that the aver-
age %CV for CD4 EQA across Trials 01-04 from
participating DP laboratories was high (18.9%) but
later dropped to less than 10%. This may be because
the program was relatively new to these participating
laboratories at the beginning of this EQA program.
Our results using this DP technique were similar to
those of Malone et al.9 (%CV range of 8.4% -23.0%)
and Goguel et al.10 (12.5%-14.5%). The average %CV
of DP for the 30 trials was 7.2%, which was higher
than the 19 SP trails (%CV of 5.6%) but lower than
Malone et al.9 and Goguel et al.10 studies.

Fig. 2 shows that %CD4+T-lymphocyte
mean value for EQA Trial 07 conducted in 2003 ex-

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231
hibited a moderate level of fluctuation. While most
values were within ± 2SD, some exceeded 2SD. In
contrast to Trial 15 (2005) in which all the same in-
dividual laboratories (Laboratory No. 1 to Laborato-
ry No. 48) performed exceedingly well with confined
within 1 SD. It is reasoned that the longer the la-
boratories participating in the EQA trial, the better
the performance of these laboratories.

Each year the EQA provider sends certifi-
cates of participation to members who are involved
in all six trials. Participation is a requirement for any
public health laboratory seeking certification for a
national17 or international18 laboratory standard. In
the near future, the EQA provider expects a new re-
porting system to be implemented. An internet-based
reporting system is being developed that will allow
all members to upload their results online. Further-
more, the designed report will be similar to laborato-
ry test results acquired using other software, and
which will be provided as longitudinal plots. This
internet-based program is less complicated than other
programs19 and will also feature additional data for
reagent tracking reports, including expiration date,
lot number and manufacturer. The provider will gen-
erate a report to be returned to individual members
within four weeks, thereby replacing the current ma-
nual report. The major benefit of this application is
that it can display data in charts, and members can
monitor their performance and compare their find-
ings with other participants.

There are two EQA country programs direct-
ly supported by NAP funding. One is the HIV Se-
rology EQA Program, provided by the National In-
stitute of Health.20 The other is this CD4 EQA pro-
gram, which is provided by the center. The latter
program has recently been integrated into the Na-
tional Health Security Office Program and, as a re-
sult, the program provider has received some funding.
Both programs have received national recognition
throughout the medical profession.

There are some limitations to this program.
Firstly, it reflects the performance of each participat-
ing laboratory in the analytical but not the pre- or
post-analytical phases, since participating laborato-
ries were aware that they were processing an EQA
sample. Therefore, the interpretation of the program
may not be direct and accurate indications of labora-
tory performance for the analysis of patient sam-
ples.21 Some laboratories may perform well with
EQA samples, but do so poorly using patient sam-
ples if they are not informed of the errors that can
occur in pre- or post-analytical phases. Indeed, a
blind EQA program might help improve routine test-
ing. Unfortunately, no such program has yet been
established in Thailand.

Secondly, there were a number of problems
found during our evaluation. Although there were
no complications with the lysis and lymphocyte gat-
ing procedures, some flow cytometers encountered
technical problems arising from inconsistent main-
tenance issues or because participants did not per-
form calibration tests prior to sample runs, leading to
inaccuracies. Our assistance was required in this re-
gard when we were contacted by participants, or dur-
ing our annual training meetings.

In conclusion, our CD4 EQA program using
stabilized blood preparations improves the reliability
of CD4+T-lymphocyte determinations. This is be-
coming increasingly important as Thailand scales up
its national ART access program for persons living
with HIV/AIDS. This national EQA program has ari-
sen despite numerous challenges, requiring almost a
decade to reach its present status.22 It may be consi-
dered a significant advancement for Thailand as well
as for the Association of Southeast Asian Nations
(ASEAN), particularly as no such program had been
available previously. Among its major roles are to
provide adequate and ongoing information to its par-
ticipants while evolving in an effort to meet partici-
pant satisfaction.17,18 In addition, the program is cur-
rently expanding to neighboring ASEAN countries
without incurring any major operating costs, thus as-
sisting neighboring nations to implement the im-
provements that we have developed in our present
study.

ACKNOWLEDGEMENTS

The authors would like to thank all partici-
pating flow cytometry laboratories, as well as the fi-
nancial support of the Global AIDS Program, CDC,
National Health Security Office, Ministry of Public
Health of Thailand, and the Thailand Research Fund-
Senior Research Scholar Award. They would also
like to thank Becton Dickinson Biosciences (Thail-
and) for technical assistance and training.

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232
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