Pattanapanyasat, Kovit PhD; Noulsri, Egarit PhD; Lerdwana, Surada BSc; Sukapirom, Kasama MSc; Onlamoon, Nattawat PhD; Tassaneetrithep, Boonrat PhD, MD

INTRODUCTION

CD4⁺ T-lymphocytes are primarily targeted by HIV and are progressively depleted during the course of the disease which eventually results in the development of AIDS.^1-3 The measurement of CD4⁺ T-lymphocyte counts is a more useful and affordable guide than plasma viral load⁴ for clinical decision making throughout the course of HIV/AIDS from disease progression assessment to antiretroviral drug therapy (ART) initiation and for monitoring ART effectiveness in guiding regimen changes and initiation of opportunistic infection prophylaxis.^3-6

Immunofluorescence analysis by flow cytometry (FCM) is the most accepted standard technology for CD4⁺ T-lymphocyte counting because of its accuracy, precision, reliability, and also the method of choice if a large throughput of samples (100-200 samples per day) is required.^7,8 Alternative non-FCM technologies, although less expensive, have not been widely used due to their complexity, low throughput (1-10 samples per day), and poor quality control.^9-11 Absolute CD4⁺ T-lymphocyte count determination by FCM may be performed using either a dual-platform (DP) or single-platform (SP) method.^7,8,12-14 The DP FCM approach estimates absolute CD4⁺ T-lymphocyte counts by a mathematical formula using 2 independent parameters; percent CD4⁺ T-lymphocytes obtained by FCM and total white blood cell count (WBC) and differential lymphocyte counts determined by a hematological analyzer or a hematocytometer. An absolute CD4⁺ T-lymphocyte count is then derived by multiplying percentage of lymphocytes that are CD3⁺/CD4⁺ by the absolute lymphocyte count. The SP FCM approach, on the other hand, enables the absolute CD4⁺ T-lymphocyte counts to be derived directly without the need for a hematological analyzer. The SP approach is based either on a volumetric principle by counting CD4⁺ T-lymphocytes in a precisely determined blood volume^15-18 or on the concept of using the known numbers of fluorescent microbeads admixed to a known volume of CD4-stained blood.^19-22 More recently, the use of flow rate of the acquiring sample during FCM analysis has been proposed.^23-25 Nevertheless, the operation and the quality control of the FCM with regard to the constant flow rate are not simple; they require substantial technical expertise and instrument maintenance. The SP bead-based FCM system is quite expensive (US $ 25-30) when compared with the DP FCM system using a hematological analyzer (US $ 15-20). An additional cost of US $ 6-8 (local price) for the reference microbeads either in the form of a lyophilized pellet or liquid suspension prevents this SP bead-based system from being widely used in many resource-limited countries. The use of SP volumetric FCM system, although simple and inexpensive and reliable, requires a new volumetric FCM, which is not available in resource-limited settings and unlikely to be installed due to the additional cost involved in these resource-limited countries. Consequently, FCMs that utilize the DP approach are still the preferred choice in many countries including Thailand and India despite many drawbacks, which include inconvenience, problems with accuracy, and above all, the wide interlaboratory and intralaboratory variability due to the use of various hematological analyzers with different methodologies.²⁶

Glutaraldehyde-fixed chicken and rainbow trout red blood cells are often used for FCM calibration or used as standards for DNA content determination.^27-29 These fixed avian and fish cell nuclei do not need any staining because they become fluorescent due to the compounds formed by aldehyde binding to amino groups in the proteins of the cells. Because they fluoresce as bright as the level of the immunofluorescently stained cells, they are often used to simulate cells stained with fluorescent antibodies. Moreover, glutaraldedyde-fixed chicken red blood cells (CRBCs) are cheap and easy to prepare. In this study, we used these autofluorescent CRBCs as the counting beads in determining the absolute CD4⁺ T-lymphocyte counts from HIV-1-infected blood samples and compared these values with those obtained by the standard fluorescent microbeads in the 3-color FCM method. Results of this study for the first time demonstrates that these glutaraldehyde-fixed CRBCs can be used as fluorescent microbeads for performing SP CD4⁺ T-lymphocyte counting that is not only a great improvement on reliability and reproducibility, but also a marked reduction in the costs involved in enumerating the absolute CD4⁺ T-lymphocyte counts in HIV-1-infected blood samples.

MATERIALS AND METHODS

Preparation of Glutaraldehyde-Fixed CRBCs

Twenty milliliters of fresh whole blood were obtained from a 14-week-old Gallus gallus domesticus chicken by venipuncture from vein of the chicken wing using a 50-mL syringe containing 5 mL of heparin solution (1000 U/mL). The chicken was kept, maintained, and treated in adherence to the guide for the Research/Teaching Proposals Involving Animal Subjects [Siriraj Animal Care and Use Committee (SI-ACUC)]. The heparinized blood was transferred to a 50-mL centrifuge tube and centrifuged at 350g for 5 minutes at room temperature (24°C to 26°C). The supernatant and the buffy coat including all white blood cells were removed using a Pasteur pipette. The CRBC pellet was washed twice with phosphate buffered saline (PBS), pH 7.4 and resuspended in 25 mL of PBS. A solution of 25 mL of 0.2% fresh prepared glutaraldehyde solution (prepared by adding 0.8 mL of 25% glutaraldehyde {Sigma, St. Louis, MO, USA} in 100 mL PBS) was added to CRBC suspension. The fixed CRBCs were incubated overnight with gentle rocking, washed once with PBS, and resuspended in 50 mL 0.1 M glycine solution (prepared by dissolving 9.8 g Sodium glycinate {Sigma} in 900 mL distilled water and adjusted to 1000 mL) at 4°C for 30 min. Following centrifugation at 250 × g for 5 min at room temperature, the fixed CRBC pellet was washed twice with PBS containing 1% sodium azide (Sigma), and resuspended in 20 mL PBS-azide solution. Finally, the cell density of this glutaraldehyde-fixed CRBC solution was determined by Guava® Personal Cell Analyzer (Guava Technologies, CA, USA) using ViaCount software (Guava), and the cell CRBC suspension adjusted to final density of 12 × 10⁶ CRBCs/mL. The glutaraldehyde-fixed CRBC solution was aliquoted into one mL each and stored between 4°C to 8°C as stocks. The number of cells within each aliquot was recounted 10 times using the Guava, and the same lot of CRBCs with the density of 11715/μL or 58575/5 μL was used throughout this study. To assess the precision and accuracy of the Guava system, an internal control set of Check beads kit from Guava was used before counting on each lot of glutaraldehyde-fixed CRBCs. For those clinical laboratories where Guava system is not available, counting the density of glutaraldehye-fixed CRBCs can be performed by using the reference microbeads [eg, TruCOUNT from Becton Dickinson Biosciences (BDB), San Jose, CA] admixed to a known volume of fixed CRBCs and count on FCM.

Patients and Blood Samples

Aliquots of peripheral blood samples from 87 HIV-1-seropositive patients were obtained from blood samples that were routinely submitted for immunological diagnosis to the Department of Immunology, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand. The blood samples used in this study were residual samples that were unlinked from identifiers, and no extra samples from patients were required. These whole blood samples were collected by venipuncture into K₃EDTA-containing tubes, kept at room temperature, and processed for immunophenotyping within 6 hours. The diagnosis of HIV-1 infection was based on results of serological testing, with confirmation by 2 other different serological tests. This study was approved by the Ethics Committee of the Faculty of Medicine Siriraj Hospital.

Monoclonal Antibodies and Reagents

The monoclonal antibodies used in this study were TriTEST 3-color monoclonal antibody reagents of CD3/CD4/CD45 obtained from BDB. The 3 monoclonal antibodies were directly conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), and peridinin chlorophyll protein (PerCP), respectively. The fluorescent-integrated TruCOUNT beads of known density in the standard 12 × 75-mm polystyrene tubes were purchased from BDB.

Immunophenotypic Staining

The Standard TriTEST/TruCOUNT Tubes Method

The staining of the peripheral blood samples were performed according to the BDB's recommendations. Briefly, 20 μL of the TriTEST reagent and 50 μL aliquots of EDTA-anticoagulated whole blood obtained by the reverse pipetting technique were added to a TruCOUNT tube. The mixture was vortexed gently and incubated for 15 minutes at room temperature in the dark before 450 μL of FACS lysing solution (BDB) was added. After 15 minutes of incubation, the lyse-no-wash-stained blood samples were analyzed with the FACSCalibur FCM (BDB).

The TriTEST/CRBC Method

Immunostaining with the TriTEST/CRBC method was performed by dispensing 20 μL of the TriTEST reagent and 50 μL of the EDTA-anticoagulated whole blood into a 12 × 75-mm polystyrene tube. All tubes were thoroughly mixed and incubated at room temperature for 15 minutes in the dark. After the incubation period, 450 μL of (1x) FACS lysing solution was added to each tube and incubated for another 10 minutes. Finally, 5 μL of CRBCs from the stock was added and vortexed gently before FCM analysis using the FACSCalibur.

Flow Cytometric Analysis

Each TriTEST reagent-stained blood sample was acquired and analyzed using MultiSET software (BDB) for the standard TriTEST/TruCOUNT tube method and CellQUEST software (BDB) for the TriTEST/CRBCs method. In the standard method, blood sample stained with FITC, PE, and PerCP-conjugated monoclonal antibodies were detected using the logarithmic amplification of green (530 nm), orange (575 nm), and red (675 nm) fluorescence, respectively. Forward scatter (FSC-H) and side scatter (SSC-H) signals were measured using a linear scale. After acquiring data on 15,000 events, the MultiSET software algorithm which automatically sets the gate for the lymphocyte cluster (SSC-H^low/CD45 PerCP^high+ cells)(R1). Cell outside this gate were considered to be monocytes (SSC-H^intermediate/CD45 PerCP^{intermediate+}) and granulocytes (SSC-H^high/CD45 PerCP^low+; Fig. 1A). The reference microbead population appeared as a bright, compact cluster with high SSC-H. Once the lymphocyte gate was established, the percentages of CD3⁺/CD4⁺ T-lymphocytes was determined within the gated lymphocyte population by their CD3 FITC/CD4 PE fluorescences (Fig. 1B). By gating the fluorescent TruCOUNT microbead population (R2) of the ungated CD3 versus CD4 plot (Fig. 1C) during analysis, absolute CD3⁺/CD4⁺ T-lymphocyte counts were automatically enumerated by comparing positive cellular events to bead events as determined by the BDB MultiSET software.

Figure 1

When a blood sample stained with the TriTEST/CRBC method was analyzed by the CellQUEST software, the FCM SSC-H/CD45 PerCP plot distinguished lymphocytes (SSC-H^low/CD45 PerCP^high+), monocytes (SSC-H^intermediate/CD45 PerCP^{intermediate+}), and granulocytes (SSC-H^high/CD45 PerCP^low+) similar to that obtained from the TriTEST/MultiSET system (Fig. 1D), except the CRBCs which showed a discrete population of low to high SSC-H and low red fluorescent expression. Despite the low PerCP expression, these CRBCs seemed as distinct clusters with good separation from the lymphocyte population, and thus enable a gate region to be set manually on the lymphocyte cluster. Once the lymphocyte gate was established, the percentage of CD3⁺/CD4⁺ T-lymphocytes were then manually generated by the quadrant analysis of CD3 FITC vs. CD4 PE plot (Fig. 1E). To enumerate the absolute CD3⁺/CD4⁺ T-lymphyocyte counts, another gate region (R2) was set around the bright CRBC cluster (Fig. 1F). The ratio of CD3+/CD4+ T-lymphocytes to the CRBC events in the R2 gate multiplied by the known concentration of CRBCs was used to calculate the absolute numbers of CD3⁺/CD4⁺ T-lymphocytes per microliter of blood using the following formula.

Quality Control and Assay Precision

Equation (Uncited)

To ensure quality control of the FCM immunophenotyping with regard to both performance of personnel and instrument, the same batch of reagents including the same lot of glutaraldehye-fixed CRBCs was used throughout the study. The FCM photomultiplier tube voltage, sensitivity, and fluorescent compensation settings were optimized before sample acquisition and analysis using Calibrite beads (BDB). In addition, a control sample from a normal subject and an internal quality control stabilized whole blood preparation from a CD-Check Plus CD4 Low (Streck, Omaha, NE) with established CD4 values for percent positive and absolute counts were also used with each run to assess system performance. FCM characteristic of SSC-H vs. CD45 PerCP dot plot from each acquiring sample was also visually inspected to determine if leucocyte populations are diffuse and whether there was little or no separation between lymphocyte and CRBCs clusters. Moreover, all of the immunostaining procedures and the FCM analyses were performed by the same operator who had experience using reverse pipetting technique.

To assess the precision of the TriTEST/CRBC method and the standard TriTEST/TruCOUNT system, absolute CD4⁺ T-lymphocyte counts from 10 replicates of 1 HIV-1-infected blood sample and 8 replicates of 1 blood sample derived from the same lot of CD-Check Plus CD4 Low were determined for assessment of the within-run and between-run variation, respectively. Within-run and between-run coefficient variations (CVs) of the 2 methods were determined.

Statistical Analysis

Validation of the CD4⁺ T-lymphocyte values obtained from the novel TriTEST/CRBC method was performed by linear regression analysis and Coefficient of determination (r²) using SPSS software (Release 10.0.5, Chicago, IL). To determine whether the new method is likely to differ from the standard TriTEST/TruCOUNT system, and that the new method agreed sufficiently to be interchanged with the standard method in practice, Bland-Altman statistical bias analysis³⁰ was used by graphically plotting the difference between each data pair of measurements (new method-standard method) on the vertical axis against the average of the pair (new method + standard method)/2 on the horizontal axis. The average absolute difference between the 2 methods (the bias) and the limits of agreement (LOA; equivalent to the mean difference ± 2 SD) were then calculated. Percent similarity which allows for a direct comparison between CD4⁺ T-lymphocyte values obtained by the 2 methods was also performed by taking the average between the 2 methods divided by the standard method and multiplying by 100.³¹ CV was also determined and used to define the relative level of agreement between the 2 methods.

RESULTS

Assay Precision

When both TriTEST/CRBC method and the TriTEST/TruCOUNT system were monitored for within-run variation by using HIV-1-infected blood sample, the mean CVs of CD4⁺ T-lymphocyte counts derived from 10 replicate samples were less than 1.5% (Table 1). For between-run reproducibility, the mean CVs of 8 replicate measurements from a CD-Check Plus CD4 Low obtained from the 2 methods analyzed over the period of 6-month study were less than 3% (Table 1).

Table 1

Stability Testing of the CRBCs

Analysis of the fluorescent characteristics of the CRBCs showed that essentially all of the CRBCs emitted green, orange, and red fluorescence (Fig. 2A-C). The mean fluorescent intensity (MFI) of the green fluorescence obtained using CRBCs was higher than the CD3 FITC signal (Fig. 2D), but the MFI noted for the orange fluorescence emission was lower than the CD4 PE signal (Fig. 2E). Although the MFI of the red fluorescent intensity of these CRBCs was lower than those from the CD45 PerCP emission, a clear separation of the CRBCs signal and the PerCP-positive lymphocyte cluster was noted (Fig. 2F). When these CRBCs were tested for their stability for a period of 6 months on the BDB FACSCalibur with the same FCM photomultiplier tube voltages and compensation levels, the MFI of the green, orange, and red emission remained constant throughout this period (Table 2). In addition, there was no detectable distortion of the FSC-H/SSC-H of these CRBCs. The density of CRBCs as determined by Guava also remained constant throughout the study period (Table 2).

Table 2

Figure 2

Effect of the CRBCs on the Standard TriTEST Staining Procedure

After the immunostaining of the patients' blood samples with the BDB TriTEST reagents, the stained samples were lysed and admixed with the CRBCs as described above. Various FCM characteristics were determined by the BDB FACSCalibur using the CellQUEST software. First of all, there was no difference in the FCM characteristics of both FSC-H/SSC-H and the MFI seen with the green, orange, and red signals obtained on appropriately stained white blood cells in the presence and absence of CRBCs. Second, the lysing properties of white blood cells after adding the CRBCs were similar to those without the CRBCs. Last, and of importance to the studies reported herein, the MFI and FCM fluorescent characteristic of CD3⁺/CD4⁺ T-lymphocytes obtained on aliquots of the stained samples in the presence or absence of CRBCs was similar if not identical.

Evaluation of the CRBCs on the Determination of CD4⁺ T-Lymphocytes

The mean percent CD4⁺ T-lymphocyte from each of the 87 HIV-1-infected blood samples obtained from FACSCalibur using the new TriTEST/CRBC method and from the standard TriTEST/TruCOUNT system showed essentially similar percent CD4⁺ T lymphocytes with excellent correlation (r² = 0.99, y = 1.02x - 0.10, P < 0.0001, Fig. 3A). No significant systematic bias was observed between the 2 methods, giving a minimum overall bias of +0.35% and a narrow LOA from -1.86% to +2.57% (Fig. 3B). The percent similarity over the full range of the percentages of CD4⁺ T-lymphocytes was 101.34% with a CV of 5.26% (Fig. 3C). These data indicate that the 2 methods yield comparable percent CD4⁺ T-lymphocyte values with excellent agreement.

Figure 3

Values for absolute CD4⁺ T-lymphocyte counts were also analyzed and shown also to be similar using the 2 methods. The correlation coefficient of these values was highly significant (r² = 0.98, y = 0.93x - 4.43, P < 0.0001, Fig. 3D). Bland-Altman plot of the agreement between the new method and the standard method gave a low bias value of −47.76 cells per microliter with LOA between −191.34 cells per microliter and +98.81 cells per microliter, Fig. 3E). The percent similarity was similarly high, with 96.13% (CV = 7.29%, Fig. 3F) when the 2 methods were compared. When the absolute CD4⁺ T-lymphocyte counts obtained by the 2 methods were compared in the range of ≤200 cells per microliter, the correlation was excellent (r² = 0.96, y = 0.95x + 0.11, P < 0.0001, N = 19, Fig. 3G). The mean bias was −4.48 cells per microliter (LOA, −31.92 cells/μL to +22.95 cells/μL, Fig. 3H) with percent similarity of +103.71% (CV = 14.06%, Fig. 3I). These results indicate that the absolute CD4⁺ T-lymphocyte counts determined by the new TriTEST/CRBC method are in good agreement with the standard TriTEST/TruCOUNT method.

DISCUSSION

According to sentinel data from the 2007 report on the Global AIDS Epidemic by the joint United Nations Program on HIV/AIDS and World Health Organization,³² the estimated individuals living with HIV worldwide was 33.2 million (30.6 - 36.1 million), with sub-Saharan Africa carrying the highest percentage of the global burden. Every day more than 6800 people living in low-income and middle-income countries become newly infected with HIV, and more than 5700 people die from AIDS, mostly because of inadequate access to HIV prevention and treatment services. Thus the introduction of inexpensive and generic ART has been an important step forward in the fight against HIV/AIDS pandemic in the resource-limited settings. However, adequate management of the health care of individuals infected with HIV is possible only if the ART program is accompanied by accessible and affordable laboratory services such as the monitoring of the percentage and absolute numbers of CD4⁺ T-lymphocytes that facilitates clinical decision making. The use of the standard “state of the art” SP bead-based FCM system, although precise, accurate, and reproducible, is more expensive than the inconvenient and less reproducible DP FCM method when the cost of the reference microbeads, that is, BDB TruCOUNT tube or Beckman Coulter (BC, Miami, FL) Flow-Count liquid bead suspension is considered. This SP FCM system using TruCOUNT tubes or BC Flow-Count is thus difficult to apply for routine use in most laboratories in resource-limited settings. This problem has prompted several manufacturing companies and investigators including our group to develop simpler and less expensive FCM methods for measuring absolute numbers of CD4⁺ T-lymphocytes.^15-25,33 We submit that the use of CRBCs as outlined in the present study will not only help reduce the inaccuracy and variability of the absolute CD4⁺ T-lymphocyte counts commonly associated with the DP FCM method widely used in the resource-poor settings, but may also prove to be beneficial to a select group of resource rich countries, where health care budgetary constraints is under increasing pressure to operate in a more cost effective manner. Furthermore, after fixation, our CRBCs have a shelf life of at least 6 months when kept at 4^oC and remain very stable even being used a lot. Of equal significance is the low cost of our CRBCs associated with the TriTEST method. Using the CRBCs to replace the commercial reference microbeads will dramatically reduce the cost from US $ 6-8 to US $ 0.2, which represents a cost reduction of 45%-50% per test based on the locally available price of the microbeads (Table 3).

Table 3

Results of the studies reported herein demonstrate that the simple glutaraldedhyde-fixed CRBC preparation can be successfully integrated into the standard TriTEST/CellQUEST FCM method or the BC EPIC XL System software. Addition of CRBCs into the TriTEST-stained blood samples led to no detectable change in FCM characteristics of FSC-H/SSC-H, SSC-H/CD45 PerCP and fluorescent pattern of CD3 FITC/CD4 PE. Although the FITC+/PE+ CRBC cluster was not as bright as the BDB TruCOUNT microbeads, they have markedly different fluorescent scatter characteristics than the CD3⁺/CD4⁺ T-lymphocyte fluorescence, thus enabling quantitation of the number of CRBCs in this cluster, which is in turn used for calculating the absolute numbers of CD4⁺ T-lymphocytes. In the present study, we have compared the percent CD4 and absolute CD4⁺ T-lymphocyte values in HIV-infected blood samples obtained by the TriTEST/CRBCs with the standard TriTEST/TruCOUNT method and found the results to be highly comparable with r² values of 0.99 and 0.98 for percent and absolute values, respectively. The overall bias for percent CD4⁺ T-lymphocyte values was +0.35%, and the similarity was 101.34%. This is no surprise as the 2 methods use the same staining TriTEST monoclonal antibody reagents and the standardized features of the FCM. These data indicate that the CRBCs pose no interference in the determination of percent CD4⁺ T-lymphocytes in HIV-infected blood samples. When comparison was made for absolute CD4⁺ T-lymphocyte values, the new TriTEST/CRBC method also showed excellent agreement with a small mean bias of about −47.76 cells per microliter. The percent similarity of 95% means that there is <5% difference in values utilizing these 2 methods.

Although this evaluation study suggests that our TriTEST/CRBC method is easy and convenient to use as it is similar to the use of liquid suspension BC Flow Count, and performs well when compared with the standard TriTEST/TruCOUNT method, there are some limitations and challenges for implementation: (1) Our TriTEST/CRBC method has to be performed using the manual CellQUEST software, a versatile software program that requires the operator to adjust the settings of the instrument and conditions for data acquisition, storage, and analysis (ie, creation of SSC-H^low/CD45 PerCP^high+ lymphocyte region and setting up of quadrant on the CD3 FITC/CD4 PE). Some operator training and experience are obviously required for using the CellQUEST software. This is in contrast to the TriTEST/TruCOUNT method which uses the MultiSET software system that provides automatic gating facility to support automated operation; (2) The other drawback of using the manual software is that this new method requires a relatively longer time to run and analyze the sample, making this method less practical for the high throughput laboratories (Table 3). However, with the operator's experience along with the help of the acquiring and analyzing template and the batch analysis menu in the CellQuest software, it is possible to substantially reduce the time spent on each sample; (3) Unlike the MultiSET software that provides an automated reporting system, the CellQUEST program requires the operator to manually calculate and then transfer the results to another software program for formatting before providing the data to the collaborating physician. There is obviously room for clerical errors during the transfer of data from one program to another. Nevertheless, running this new TriTEST/CRBC method on the CellQUEST software is based on the same gating hierarchy using the automated MultiSET software. Further modification of the manual software can be achieved by the development of a user-defined automated software based on an algorithm system.³⁴ This user-defined software will not only reduce errors obtained utilizing manual gating but will also help increase laboratory throughput from 25 samples per hour to 30 samples per hour as in the standard automated TriTEST/TruCOUNT method; (4) There are 4 pipetting steps in the TriTEST/CRBC method, whereas only 3 pipetting steps are required in the standard TriTEST/TruCOUNT method. This is because the TruCOUNT is in the form of a lyophilized pellet, which is already present in the staining tube. It is known that systems using the SP method require a higher level of precision in the dispensing of reagents in all the pipetting steps.^35,36 Thus, the addition of pipetting steps could produce more variation in the results. It is therefore reasoned that adequate training on the use of reverse pipetting technique is essential to avoid such variations; (5) Finally, because this evaluation was a proof-of-concept study and was performed in a single laboratory with the resources and technical skills that fulfill all required FCM standards, a more complete comparison, involving intralaboratory and interlaboratory evaluation should be conducted to assess the effect of typical types of variation encountered in clinical FCM laboratories before implementation of such method country wide.

In conclusion, this new TriTEST/CRBC method is a valid and promising method for performing SP absolute CD4⁺ T-lymphocyte counts with excellent comparability to the standard bead-based method. We have demonstrated that this new glutaraldehyde-fixed CRBCs can easily be integrated for use as substitute microbeads for the routine FCM SP method which will markedly reduce the cost of CD4 testing without sacrificing accuracy and precision, thus potentially allowing more HIV/AIDS patients access to CD4 monitoring in resource-limited settings.

ACKNOWLEDGMENTS

We sincerely thank Professor Aftab A. Ansari for valuable discussions. We also thank National Health Security Office, Ministry of Public Health, Thailand, and Becton Dickinson Biosciences (Thailand) for additional financial support and contribution of reagents and training. Special appreciation is also extended to Charin Thepthai for assistance in the collection of patients' blood samples.

REFERENCES