Comparison of CD4 T lymphocyte in Paraformaldehyde-based Fixation and WU-Met
CD4 QC© Preparation Prototype
Issara Prachongsai1,*, Jiraporn Jaroenpool1, Egarit Noulsri2 and Kovit Pattanapanyasat2,#
1
School of Allied Health Sciences and Public Health, Walailak University, Thailand.
2
Centre of Excellence for Flow cytometry, Mahidol University, Thailand.
* issara.pa@wu.ac.th, # kovit.pat@mahidol.ac.th
Abstract
CD4 T lymphocyte is one of crucial parameter used for determining immune status in HIV
infected individuals. Flow cytometry is presently the gold standard method for CD4 T
lymphocyte enumeration [1]. Thereafter, the use of quality control materials is required to
ensure the precise and accurate parameters obtained by CD4 testing laboratories. Stabilized
whole blood has been recommended to achieve all of process during analysis [2].
Paraformaldehyde-based (PFA) fixation has been employed for blood stabilization. However,
methanol which is non-paraformaldehyde agent has been reported in its preservation on
cellular morphology when followed by paraformaldehyde fixation [3]. WU-Met CD4 QC© is
fixative agent being patented by Walailak University which is formulated with nonparaformaldehyde agents. Peripheral blood obtained from healthy volunteer collected in
K3EDTA were stabilized with various concentration of WU-Met CD4 QC© and PFA fixation
was used as positive control. CD4 T lymphocyte percentages and absolute counts were
obtained from WU-Met CD4 QC© by flow cytometry and compared to PFA fixation at
different time points. The investigation showed that the optimal condition for blood
stabilization using WU-Met CD4 QC is 2 hours of incubation at 4 C°. The results
demonstrated that both CD4 T lymphocyte percentages and absolute counts on WU-Met CD4
QC© were stable for 30 day post stabilization. Moreover, the lesser deviation of percent
differences on absolute CD4 T lymphocyte observed in WU-Met CD4 QC© stabilization
compared to that of conventional preparation at both 4 C° and ambient temperature. The
investigation showed that this prototype agent exhibited long term stabilization and
potentially being developed for blood stabilization which is useful for daily and longitudinal
QC materials for flow cytometry CD4 testing.
Keywords: CD4 T lymphocyte, stabilized whole blood, flow cytometry, quality control
Introduction
CD4 T lymphocyte is the major target cell of human immunodeficiencies virus (HIV).
Declining of this cell type results in susceptible to opportunistic infections which are life
threatening situation in HIV infected patients [4]. World Health Organization (WHO)
recommended using CD4 T lymphocyte parameters to monitor immune status of infected
individuals [5]. Hence, CD4 T lymphocyte enumeration should be available for clinical
decision, not only for starting anti retroviral drug therapy (ART) but also monitoring the
possibility of drug resistance which usually occur in long term ART treatment [6].
Nowadays, there are many sophisticate technologies available for CD4 enumeration.
However, flow cytometry (FCM) is still accepted as gold standard method for CD4 T
lymphocyte testing [3,7]. Although FCM demonstrated precise, accurate and reproducible
results on cell analysis, the recruitment of trained person, high-cost of reagents and control
materials affected in accessibility of the quality assurance system in resource-constraint areas.
Center of Excellence for Flow Cytometry, Siriraj Hospital, Mahidol University and Ministry
of Public Health (MOPH) are the main anchors responsible for quality assessment scheme on
CD4 T lymphocyte testing in Thailand [8]. Providing stabilized whole blood preparation
supports the expense of each laboratory on quality control supplying. Although various kinds
of lyophilized white blood cells were commercial available, using stabilized whole blood that
mimic the homogeneity of blood sample has been preferred to use in quality control [2].
However, the transportation of stabilized blood usually causes degradation and integrity of
the materials. Especially, the changing and delaying of delivery system are still seriously
issues on the stability of control material [9]. Nowadays, optimal concentration of
paraformaldehyde is widely used for blood stabilization. Other non-paraformaldehyde agent
such as methanol and acetone had been introduced for alternative stabilization substances
[10,11]. Previous studied by Police AA. and colleagues revealed that the combination of
paraformaldehye with methanol enhanced preserving ability on cell morphology and other
components of blood cells[3]. Thereafter, the use of other non-paraformaldehyde agent
formulated in stabilized blood preparation may enhance the performance of new stabilizing
agents and be selected as prototype for further developing to a novel blood stabilization in the
aspect of CD4 T lymphocyte testing.
Methodology
Monoclonal antibody
The monoclonal antibodies (MAb) used in this study were purchased from BD Biosciences (BDB). These monoclonal antibodies were CD3-FITC, CD4-PE and CD45PerCP which were mixed as cocktail monoclonal antibodies (TriTEST™). For isotype
control IgG1-PE and IgG1-FITC were used. The staining procedure was performed according
to manufacturer’s instruction.
Stabilized whole blood preparation
The ethical consideration was approved by the ethical committee, Walailak
University. Peripheral blood was collected from healthy volunteers using K3EDTA as
anticoagulant. Plasma was removed by centrifugation at 2,500 rpm for 10 minutes. The pellet
was resuspended with 1X PBS twice and then fixed with various concentrations of WU-Met
CD4 QC©. Fixed whole bloods were incubated for 2 hours and overnight at 4 C°.
Paraformaldehyde-based fixation was used as positive control. The fixative agents were
removed and washed twice with 1X PBS. The pellet was doubled with collected plasma.
Sample staining
Stabilized blood was stained using lyse-wash technique. Ten microliters of stabilized
blood were mixed with 50 μL of MAb, vortex and incubated at ambient temperature for 15
minutes in the dark. RBC was lysed with 1 mL of 1x FACSlysing (BDB) for 15 minutes.
Lysing whole blood was centrifuged at 1,600 rpm for 5 minutes and followed by washing
once with 2 mL 1XPBS. The cell pellet was resuspended with 350 μL of 1%
paraformaldehyde prior analysis.
Flow cytometric analysis
FACSCalibur instrument was calibrated with CaliBRITE™ bead and FACSComp
software (BDB). Forward scatter (FSC) and side scatter (SSC) dot plot was created to define
three main subpopulations of white blood cell. Lymphocyte was gated low SSC and high
CD45 signal intensity manner. Defined lymphocytes were logically selected to analyze
frequency of CD4 T-lymphocytes on CD3 vs. CD4 dot plot. A minimum of 15,000 events of
lymphocytes were collected. Data were acquired and analyzed using CellQuest Pro™
software (BDB).
Complete blood count
Hematologic parameter including white blood cell count and differentiation, red blood
cell indicies and platelet count obtained by Nihon Kohden™ (MEK-8222). Measurement
were followed instrument’s instruction , calibration, quality control was set up prior any
testing.
Absolute CD4 T lymphocyte calculation
Lymphocyte percentage and WBC count were used to calculate for absolute CD4 T
lymphocyte count. Follow by the formula; absolute CD4 T lymphocyte count = (WBC count
x Lymphocyte percentage x CD4 T lymphocyte percentage) / 10,000
Statistical analysis
CD4 T lymphocyte percentage and absolute count were observed and collected for 30
day post stabilization. Data were analyzed and graphed using Microsoft Office Excel 2007.
Results
Optimization of Stabilization using WU-Met CD4 QC
WU-Met CD4 QC© at various concentrations and time of stabilization was tested for
characterizing the effect of fixative agent on blood stabilization. FSC/SSC dot plot was set up
to observe the integrity of WBC after stabilization. When utilized 1X WU-Met CD4 QC© ,
there was precipitant which was detected by visual observation. The integrity of WBC was
defined at 0.25x WU-Met CD4 QC (figure1). Long-term exposure of this agent would
degrade WBC integrity compared to 2 hour of stabilization. Hence, the optimal condition of
this prototype fixative agent was 0.25X with 2 hours of incubation.
Figure1. Light scatter parameter (FSC. vs. SSC.) of WBC stabilized
by 0.25X WU-Met CD4 QC© at 2 hours (left) defined discrete lymphocyte, monocyte and
granulocyte compare to those of overnight incubation (right)
Stability of CD4 T lymphocyte percentage
Stabilized bloods were stored at different conditions (4 C° and ambient temperature).
CD4 T lymphocyte percentages were acquired from stabilized blood preparation at different
time points. Percent differences were calculated by formula [(A-B)/B x 100] where A was
CD4 percentage calculated on day 0 of stabilization, B was parameters acquired on day 7,
day 14 or Day 30. The results showed that a small deviation of percent differences on CD4 T
lymphocyte percentage observed in PFA fixation and WU-Met CD4 QC© stored at 4 C°
(figure 2) while larger deviation of percent differences (>10%) were presented in PFA-based
stabilized whole blood.(figure 3)
Figure2. Bar plot representative percent differences of CD4 T lymphocyte percentages in
stabilized whole blood stored at 4 C° prepared by different stabilizing substances
at day 0 vs. day 7, day 0 vs. day 14 and day 0 vs. day 30.
Figure3. Bar plot representative percent differences of CD4 T lymphocyte percentages in
stabilized whole blood stored at ambient temperature prepared by different stabilizing
substances at day 0 vs. day 7, day 0 vs. day 14 and day 0 vs. day 30.
Stability of absolute CD4 T lymphocyte counts
Absolute CD4 T lymphocyte has been widely used compare to its percentage. The use of
WU-Met CD4 QC© to obtained stable absolute CD4 T lymphocyte counts was tested. Percent
differences of absolute count were calculated as described. Although absolute CD4 T
lymphocyte obtained by stabilized whole blood treated with PFA and stored at 4 C° exhibited
accepted range of absolute CD4 T lymphocyte count (not exceed 10% differences) (figure 4),
there was larger deviation of absolute counts in PFA-fixed stabilized whole blood which is
stored at ambient temperature.(figure 5)
Figure4. Bar plot representative percent differences of absolute CD4 T lymphocyte counts
in stabilized whole blood stored at 4 C° prepared by different stabilizing substances
at day 0 vs. day 7, day 0 vs. day 14 and day 0 vs. day 30.
Figure5. Bar plot representative percent differences of absolute CD4 T lymphocyte counts
in stabilized whole blood stored at ambient temperature prepared
by different stabilizing substances
at day 0 vs. day 7, day 0 vs. day 14 and day 0 vs. day 30.
Discussion and Conclusion
CD4 T lymphocyte parameters raised their important on HIV management. There was
essentially to obtained accurate, precise and reliable results by laboratories. Stabilized whole
blood which mimics the homogeneity of blood sample has been proposed to use as quality
control material provided by quality control schemes. However, the development of a new
stabilizing agent and process is still needed to improve the efficiency of current stabilized
whole blood preparation [12]. A new blood stabilizing agent was tested for basic
performances on its preserving ability including WBC integrity, CD4 T lymphocyte
percentages and absolute counts. The investigations suggested that there is limited range of
incubation period promotes preservation of blood cells. The robustness was observed on
some subpopulations of WBC at high concentration of stabilizing agents have been used.
Increase incubation period of stabilization resulted in the loss of WBC integrity even at
optimal concentration of blood stabilizing agent. The use of optimal concentration of nonparaformaldehyde agent formulated in WU-Met CD4 QC© agent improved the deviation of
absolute CD4 T lymphocyte counts which has been observed in PFA-based fixation. CD4 T
lymphocyte parameters stabilized in this new agent were intact even in the ambient
temperature raised the impact of stabilization efficiency. Especially, the improvement on the
variations of absolute CD4 T-lymphocyte counts which usually occur among laboratories
base on dual-platform, methods of test and instruments [2,12]. Our WU-Met CD4 QC©, a
prototype fixative agent may further develop as alternative methods as needed for stabilized
whole blood preparation.
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Acknowledgements: This work was funded by institute of research and development (IRD),
Walailak University and in cooperation with Centre of Excellence for flow cytometry (COE),
Mahidol University.