Several CD4 lymphocyte enumeration methods have been developed to be used as alternatives to conventional flow cytometry. However, no consensus has been reached on which technique should be widely adopted. We introduce here a novel nonflow cytometric method, called CD4 Select, for enumeration of CD4 lymphocytes. Instead of using a flow cytometer, the method requires only an automatic hematoanalyzer (complete blood count [CBC] machine). The correlations of the percentage of CD4 lymphocytes and absolute CD4 counts with the CD4 Select method and standard flow cytometry were 0.932 and 0.922, respectively. The CD4 Select method yielded percentage of CD4 lymphocytes greater than flow cytometry by a mean of 0.06%, and the limits of agreement at 95% confidence interval (CI) were 9.46% to −9.56% using Bland-Altman analysis. The CD4 select method is cheap, valid, and simple enough to be conducted locally at small facilities. The method yields not only absolute CD4 lymphocytes, but also percentage of CD4 lymphocytes. The use of a hematoanalyzer also gives this method relatively high sample throughput and makes it less labor-intensive than other nonflow cytometric methods.
From the *Department of Community Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; †Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand; ‡Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand; and §Biomedical Technology Research Center, National Science and Technology Development Agency at the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
Received for publication June 4, 2007; accepted November 15, 2007.
This study was supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC) of the National Sciences and Technology Development Agency and the Thailand Research Fund.
Correspondence to: Watchara Kasinrerk, PhD, Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand (e-mail: firstname.lastname@example.org); or Thira Sirisanthana, MD, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand (e-mail: email@example.com).
Highly active antiretroviral treatment (HAART) is becoming more affordable and accessible for persons living with HIV in developing countries.1 Promotion of HIV testing and counseling has also resulted in more HIV-infected persons seeking care from the health service system long before their CD4 lymphocyte counts reach the levels that require HAART initiation. A survey among young adults in northern Thailand found that nearly half of them had already been tested for HIV.2 To provide clinical care for persons living with HIV, whether or not they are currently on HAART, the clinician must routinely monitor their CD4 lymphocyte levels.3 Quality monitoring of CD4 lymphocytes in HAART patients also reduces the future need of other high-cost interventions, such as a more expensive medication regimen and drug resistance test. However, flow cytometry, the gold standard for CD4 lymphocyte enumeration, is too expensive and too complicated to be practical in resource-limited settings.4 Alternative methods to determine CD4 lymphocyte levels are needed.
Ideally, the alternative methods should yield accurate and reliable results comparable to the standard flow cytometric method. The cost of initial laboratory setup and per-sample running cost should be minimal. The method should also be easy to perform and not need high-level technical skill and extensive staff training. To decentralize the care of persons living with HIV from sophisticated medical centers to community hospitals near the patients' homes, the number of tests to be run at a time should be flexible and not need to be done in batch. Because of the long turnaround time required for the standard flow cytometric method, the patient usually has to visit the health care facility twice for each CD4 lymphocyte test. The first visit is for the blood drawing and the second for seeing the physician when the CD4 count result is ready. This creates inconvenience and increases travel cost. To solve this problem, the candidate method should provide same-day test results.
Several CD4 lymphocyte enumeration methods, both flow and nonflow cytometric, have been developed as alternatives to conventional flow cytometry. However, no consensus has been reached on which technique should be used, and none has been widely adopted.4,5
In our previous report, we introduced and evaluated a simple nonflow cytometric CD4 lymphocyte enumeration method called the MT4 rosetting method.6 In that technique, inert latex beads were coated with a CD4 monoclonal antibody (mAb) named MT4, which was generated in our laboratory. The MT4 mAb strongly recognizes CD4 protein on CD4 lymphocytes but reacts weakly or not at all with CD4 protein on monocytes.6 The MT4 rosetting reagent was mixed with the patient's blood, and white blood cells having 3 or more latex particles attached to them were counted under a visible light microscope. We concluded that our MT4 rosetting method was inexpensive, easy to perform, and correlated well with the standard flow cytometric method.
Because CD4 lymphocytes have to be manually differentiated from other cells, the MT4 rosetting method still has some limitations when many samples have to be tested at the same time. Realizing the limitations of the MT4 rosetting method and the advantageous characteristics of MT4 mAb that only react with CD4 on CD4 lymphocytes, in this article we introduce a novel, simple method for enumeration of CD4 lymphocytes, called CD4 Select. The method is simple, inexpensive, and accurate. It only requires an automatic hematoanalyzer (complete blood count [CBC] machine), which is already available in most hospitals' laboratories. The CD4 Select method is therefore an alternative for enumerating CD4 lymphocytes to assess HIV/AIDS patients, particularly in small health care facilities.
MATERIALS AND METHODS
Whole blood samples were obtained from 100 HIV-infected individuals using Vacutainer tubes (Becton Dickinson Biosciences, San Jose, CA) containing tripotassium ethylenediaminetetraacetic acid (K3EDTA) at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand. The study subjects were randomly selected in a blinded manner. Blood samples were processed for CD4 lymphocyte count within 6 hours of collection. The study was approved by the Chiang Mai University ethical committee.
Production and Purification of CD4 mAb MT4
Hybridoma clone MT4, which is a CD4 mAb immunoglobulin M (IgM) isotype, was generated from a mouse immunized with the SupT1 human T-cell line by standard hybridoma techniques.7 To produce high concentrations of mAb, the MT4 hybridoma clone was injected intraperitoneally into Balb/C mice that were pretreated with 2, 6, 10, 14-tetramethylpentadecane (Pristane; Sigma, St. Louis, MO). Ascites containing CD4 mAb were harvested, usually 2 to 3 weeks after hybridoma inoculation. The mAb MT4 was purified from the induced ascites by affinity chromatography using a HiTrap IgM purification HP column (GE Healthcare Bio-Sciences, Piscataway, NJ).
Flow Cytometry for Enumeration of CD4 Lymphocytes
To enumerate CD4 lymphocytes by flow cytometry, a 2-color, dual-platform, flow cytometric method was employed. Briefly, 100 μL of K3EDTA whole blood were incubated at room temperature with 20 μL of each reagent panel (Simultest; Becton Dickinson Biosciences) in separate tubes. The reagent panel was composed of 4 2-color reagent pairs: leukoGATE (CD45-FITC/CD14-PE), control IgG1-FITC/IgG1-PE, CD3-FITC/CD4-PE, and CD3-FITC/CD8-PE. After 15 to 30 minutes incubation at room temperature, 2 mL of lysing solution (Becton Dickinson Biosciences) was added, and the samples were allowed to stand at room temperature in the dark for 10 minutes. Cells were then washed once with phosphate-buffered saline containing 0.1% sodium azide. Samples were subsequently fixed with 1% paraformaldehyde and analyzed by a flow cytometer with Simultest IMK-Lymphocyte software (Becton Dickinson Biosciences). The percentages of CD4 lymphocytes in the total lymphocyte population were determined via the Simultest software. The absolute CD4 lymphocyte count was then calculated from the total white blood cell count and the percentage of lymphocyte subpopulation.
Preparation of CD4 Select Reagent
Ferrous beads (Dynalbeads M-450 Eproxy; Dynal Biotec, Oslo, Norway) were washed 3 times with 0.1 M phosphate buffer pH 7.4 (PB) using a magnetic particle concentrator (MPC; Dynal Biotec). The beads were then adjusted to 5.0 × 107 beads/mL with PB. Purified mAb MT4 100 μg was added to 1 mL bead suspension. The mixture was rotated at room temperature for 1 hour. Then, the beads were pelleted by a magnet and 500 μL of PB were removed. Five hundred μL of 2% bovine serum albumin in phosphate-buffered saline containing 0.02% sodium azide (2% BSA-PBS-0.02% NaN3) were added. The beads were rotated at 4°C for 16 to 20 hours. The beads were then washed 3 times with phosphate-buffered saline. The washed beads were adjusted to 1.0 × 107 beads/mL in 1% BSA-PBS-NaN3. This reagent, named CD4 Select, was stored at 4°C.
CD4 Select Method for Enumeration of CD4 Lymphocytes
Two hundred μL of K3EDTA whole blood were pipetted into 2 separated tubes, tube A and tube B. Two hundred μL of phosphate-buffered saline were added into tube A and 200 μL of CD4 Select reagent were added into tube B. The tubes were rotated at room temperature for 30 minutes. The tubes were then placed on a magnetic device for 5 minutes for depletion of CD4 lymphocytes. Using a plastic transfer pipette, the blood containing the nonferrous bead-bound cells was transferred to a fresh tube. The collected blood was then subjected to analysis by an automatic hematoanalyzer. The percentage of CD4 lymphocytes in total lymphocyte was then calculated by the equation
where %L1 and %L2 are the percentage of lymphocytes from tube A and tube B, respectively.
From the obtained percentage of CD4 lymphocytes, the absolute CD4 lymphocyte count was then calculated from the total white blood cell count and the percentage of the lymphocyte subpopulation.
Correlation coefficients were used to compare the CD4 lymphocyte counts obtained from standard flow cytometry and the CD4 Select method. A Bland-Altman plot8 was used to analyze agreement between the 2 methods.
A CD4 mAb named MT4 was produced in our laboratory.6,7 This mAb was reported to react with CD4 expressed on CD4 lymphocytes but reacts weakly or not at all with monocytes, which also express CD4 molecules.6 To further confirm the specificity of mAb MT4, peripheral blood cells from 9 different donors were stained with mAb MT4 and analyzed by flow cytometry. We could confirm the previous finding that in all subjects, CD4 lymphocytes were strongly positive with mAb MT4. Monocytes showed negative or very weak reactivity to mAb MT4 (data not shown). We, however, have no experimental results that bear on the question of why mAb MT4 fails to bind to monocytes.
As mAb MT4 reacts specifically react with CD4 lymphocyte, this mAb is advantageous for development of any method for enumeration of CD4 lymphocytes. In this study, purified mAb MT4 was coated on ferrous beads. After bead coating at the optimal concentrations of mAb MT4 (100 pg/mL) and beads (5.0 × 107 beads/mL), all beads showed positive reactivity with fluorescein isothiocyanate-conjugated antimouse immunoglobulin antibody (Fig. 1), indicating that all beads were coated with mAb MT4. The MT4-coated ferrous beads (named CD4 Select reagent) were then incubated with whole blood sample. In the reaction tube, the beads will bind to CD4 lymphocytes. By the principle of negative cell depletion, CD4 lymphocytes should be depleted from the blood sample after being incubated with CD4 Select reagent in a magnetic field. CD4 lymphocyte-depleted blood was analyzed by an automatic hematoanalyzer and the percentage of lymphocytes was obtained. In parallel, the same blood sample, but without addition of CD4 Select reagent, was also analyzed for the percentage of lymphocytes. The percentage of lymphocytes obtained from both analyses were used to determine the percentage of CD4 lymphocytes in the lymphocyte population by the formula described previously.
To validate the accuracy of the new method, CD4 lymphocytes from 100 HIV-infected individuals were determined by this method and by standard flow cytometry. As shown in Figure 2, high correlation between the CD4 select method and that of the standard flow cytometry for both the percentage of CD4 lymphocytes and absolute CD4 count was obtained (correlation coefficient [r] = 0.932 and 0.922, respectively). The Bland-Altman analysis was also used to compare the percentage of CD4 lymphocyte values generated by the CD4 Select and flow cytometric methods. The CD4 select method yielded a mean percentage of CD4 lymphocytes slightly greater than that of flow cytometry (0.06%), and the limits of agreement at 95% confidence interval (CI) were 9.46 to −9.56% (Fig. 3).
The CD4 Select, a novel nonflow cytometric CD4 lymphocyte enumeration method, is based on the CD4 mAb named MT4 developed in our laboratory. The word “Select” is used to highlight the unique and advantageous characteristics of the mAb MT4, which reacts only with CD4 on lymphocytes, but not on monocytes. The MT4-coated beads bind only to CD4 lymphocytes,6 thus avoiding the laboratory step of differentiating monocytes from CD4 lymphocytes that other nonflow cytometric CD4 lymphocyte enumeration methods require.9,10
The idea of acquiring a count of CD4 lymphocytes by subtracting the number of lymphocytes in CD4-depleted blood from the number of lymphocytes in untreated whole blood as used in this method is straightforward and easy to understand. Unlike other available nonflow cytometric CD4 lymphocyte enumeration methods, which yield only absolute CD4 lymphocyte counts, the CD4 Select method can provide both the percentage of CD4 lymphocytes and absolute CD4 lymphocyte count. The percentage of CD4 lymphocytes is also an important marker. Pediatricians usually need to know the patients' percentage of CD4 lymphocytes to make clinical decisions concerning ART treatment.11
Laboratory turnaround time for this CD4 Select method is less than 1 hour. The doctors can request the laboratory test and get the result back on the same day. This short time required was achieved because of 2 factors. First, as mentioned above, the techniques used are quite simple and need little time to process. Second, a hematoanalyzer is used to automatically differentiate the types of white blood cells. The use of hematoanalyzer also gives our method high throughput and makes it less labor-intensive than other nonflow cytometric CD4 lymphocyte enumeration methods. These characteristics are very helpful and suitable for small and midsize health care facilities such as community hospitals. Automatic hematology analyzers are already available in all community hospitals and other small and midsize medical centers worldwide.
The CD4 Select method has demonstrated results comparable to the flow cytometric method for both the percentage of CD4 and absolute CD4 count. The flow cytometric method has been known as a gold standard for enumeration of CD4 lymphocyte. Nonetheless it is widely known that the method itself yields slightly variable results. Discrepancies between the 2 methods is considered to be acceptable and therefore do not necessarily indicate problems with our method. The standard flow cytometric assay is expensive. Over the past decade, several low-cost cytometric reagents for enumeration of CD4 lymphocytes have been developed. The cost of the CD4 Select ($7) is similar to those low-cost reagents, eg, Guava Easy CD4/CD8 ($7.50),5 Partec CyFlow ($5),5 and PointCARE system ($10). In addition, the CD4 Select has some advantages over the low-cost cytometric alternatives. The modified flow cytometric methods have high initial equipment cost, but the CD4 Select requires only already available CBC instrument. The CD4 Select provides both percentage and absolute CD4 count, whereas some flow-based assays (eg, Guava Easy CD4/CD85) provide only absolute CD4 count.
With more HIV-infected patients under HAART treatment in resource-constrained countries, a CD4 lymphocyte enumeration method that is cheap, valid, less labor- and time-consuming, and simple enough to be conducted locally at smaller facilities is needed. In this article, we have introduced a novel nonflow cytometric CD4 lymphocyte enumeration method that meets all of the above criteria.
We are thankful to Dr. Sawitree Chiampanichayakul for technical assistance. We thank Dr. Dale E. Taneyhill for proofreading the manuscript.
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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
CD4 counts; CD4 lymphocytes; flow cytometry; nonflow cytometric method