CD4 and CD8 enumeration for HIV monitoring in resource-constrained settings

CD4 and CD8 Enumeration for HIV Monitoringin Resource-Constrained Settings

Xiao Li,1 Christian Breukers,1 Aurel Ymeti,1 Bjorn Lunter,1

Leon W. M. M. Terstappen,1* and Jan Greve1

1University of Twente, Faculty of Science and Technology, Medical Cell Biophysics,Drienerlolaan 5, 7500AE Enschede, The Netherlands

Background: We developed a volumetric single platform image cytometer (SP ICM) that is dedicated tocount CD41 and CD81 T lymphocytes for HIV monitoring in resource-constrained settings. The instrumentwas designed to be low-cost, yet reliable, easy-to-use, and robust.Methods: Whole blood is incubated with CD3-magnetic nanoparticles, CD4-phycoerythrin (PE), and

CD8-peridinin-chlorophyll-protein complex (PerCP). The CD3 cells are immunomagnetically attracted toan analysis surface, where fluorescence images of CD41 and CD81 T lymphocytes are recorded and ana-lyzed, respectively. We compared CD4, CD8 counts, and CD4/CD8 ratio obtained by the SP ICM withthose from a SP flow cytometer (FCM) tetraCXP method on blood samples from 145 patients.Results: Good correlations were obtained (R: 0.96–0.99) between the SP ICM and the SP FCM. There

was �10% CD8 undercount in the SP ICM, which could be partly caused by CD81dim T lymphocytes thatwere not detected by the instrument or not counted by the image analysis due to the cross-talk from theCD4-PE signal in the CD8-PerCP image.Conclusions: The SP ICM is a good candidate for HIV monitoring in point-of-care settings of resource-

constrained countries. q 2008 Clinical Cytometry Society

Key terms: single platform; image cytometer; CD4 and CD8 enumeration; HIV monitoring; immunomag-netic separation; immunofluorescent labeling; point-of-care

How to cite this article: Li X, Breukers C, Ymeti A, Lunter B, Terstappen LWMM, Greve J. CD4 and CD8enumeration for HIV monitoring in resource-constrained settings. Cytometry Part B 2009; 76B: 118–126.

Since the official date for the beginning of the AIDSepidemic in 1981 in Los Angeles (1), HIV infection hasbecome a global pandemic major health emergency. Thisepidemic has formed a serious, and in many countries,devastating, crisis. It is estimated that, in 2006, 39.5 mil-lion human beings were living with HIV, among them�95% were living in developing countries (2).

Immunophenotyping of T lymphocytes subsets pro-vides important information of patients’ immune system.T lymphocytes (CD31 T lymphocytes) comprise twovarieties: Helper T cells (CD41 T lymphocytes) and cyto-toxic T cells (CD81 T lymphocytes) (3). HIV infects pri-marily CD41 T lymphocytes and kills them (4,5). HIVinfection is associated with a progressive decrease of theCD41 T lymphocytes count and an increase in viral load(3,5). CD81 T lymphocytes count increase at the begin-ning of HIV infection and keep on increasing through

the progression (6,7). For HIV-infected patients, routinemonitoring of CD41 T lymphocytes count provides im-portant information on their immune status. The CD41

T lymphocytes counts are essential to decide when tostart the antiretroviral therapy (<350 cells/ll), to moni-tor the effect of this treatment, and to decide when tostop opportunistic infection prophylaxis (8–10).

*Correspondence to: Leon WMM Terstappen, University of Twente,Faculty of Science and Technology, Medical Cell Biophysics Group, Build-ing Zuidhorst, Drienerlolaan 5, 7500 AE Enschede, The Netherlands.E-mail: [email protected]

Received 6 May 2008; Accepted 6 July 2008Published online 29 September 2008 in Wiley InterScience (www.

interscience.wiley.com).DOI: 10.1002/cyto.b.20445

Grant sponsor: STW, The Dutch Technology Foundation; Grant num-ber: TGT.6146.

Cytometry Part B (Clinical Cytometry) 76B:118–126 (2009)

q 2008 Clinical Cytometry Society

Currently, flow cytometry (FCM) is the most widelyaccepted method for CD41 T and CD81 T lymphocytesenumeration. However, FCM instruments are expensive(US$20,000–125,000), and the price of an FCM assay(US$ 5–50) is relatively high. Furthermore, the operationand maintenance of an FCM require well-trained techni-cians and stable electricity. Affordable, simple, easy-to-use,and reliable systems for CD41 and CD81 T lymphocytesenumeration are therefore urgently needed (11–13).

Previously, we developed a simple single platformimage cytometer (SP ICM) to enumerate CD41 T lym-phocytes after immunomagnetic selection and immuno-fluorescent labeling (14,15). The instrument is easy-to-use, compact, and yet reliable. It is computer-controlledand can operate on a 12-V rechargeable battery. Onlyone reagent’s incubation step is needed for sample prep-aration, which makes training easy.

Further development of the SP ICM permitted thedetection of both CD41 T lymphocytes and CD81 T lym-phocytes as well as the Helper/cytotoxic T lymphocytesratio (CD4/CD8 ratio) (15). In this system, CD3 immuno-magnetic selection is combined with CD4-phycoerythrin(PE) and CD8-peridinin-chlorophyll-protein complex(PerCP) labeling. First, CD3 cells in whole blood areimmunomagnetically attracted to an analysis surface.Then, CD41 T lymphocytes and CD81 T lymphocytescan be counted in fluorescence images. With this infor-mation, the CD4/CD8 ratio can be determined. In thiswork, we evaluated the system for its ability to countCD41 T and CD81 T lymphocytes and to determine theCD4/CD8 ratio on blood specimens of 145 patients. Theresults were compared with those obtained by the SPFCM tetraCXP method.

MATERIALS AND METHODS

Blood Collection

Blood samples from 95 randomly selected HIV2

patients and 50 HIV1 patients were supplied by MSTHospital, Enschede, The Netherlands. All blood sampleswere collected in sterile K3EDTA blood collection tubes(BD Biosciences, USA) and processed within 8 h afterthey are drawn.

CD41 T and CD81 T Lymphocytes Enumeration by SinglePlatform FCM

TetraCXP method. Samples were prepared accordingto the manufacturer’s recommendation (BeckmanCoulter, USA) (16). For samples of infants younger than2 years old, 25 ll of whole blood instead of 100 ll wasused. The samples were analyzed on a Cytomics FC500using tetraCXP software (Beckman Coulter, USA), andabsolute CD3, CD4, and CD8 counts were obtained.

CD41 T and CD81 T Lymphocytes Enumeration by SP ICM

One-step immunolabeling. To 100-ll (25 ll wasused for blood samples from children) EDTA-anticoagu-

lated whole blood, 10 ll of reagents cocktail, which con-tains 3 ll of 0.655 mg/ml CD3-ferrofluid (CD3FF, clone:CRIS-7, isotype: mouse IgG2a-j, Immunicon, USA), 4 llof 12.5 lg/ml CD4-PE (clone: RPA-T4, isotype: MouseIgG1-j, BD Pharmingen, USA), and 3 ll of 6.25 lg/mlCD8-PerCP (clone: SK1, isotype: Mouse IgG1-j, BD Bio-Science, USA) were added and mixed. After 15-min incu-bation, the sample was diluted with system buffer to afinal volume of 400 ll. Approximately 340 ll of the sam-ple solution was transferred into the analysis chamber.The chamber was plugged and placed into a magnet as-sembly (MagNest1; Immunicon, USA). After 20-min mag-netic separation, the sample was ready to be analyzed.

Instrument. During the magnetic separation period,the CD3 immunomagnetically labeled cells are subjectedto a homogeneous magnetic force pointing in the posi-tive Z-direction of the magnetic chamber (17–19). Thecells then move to the analysis surface at the upper glasssurface of the chamber. For excitation of PE and PerCP,two 3-W light-emitting diodes (LEDs) (496 nm, Lumileds,Luxeon, USA) are mounted symmetrically above the mag-net. In front of each LED, a 550-nm short pass filter(Omega Optical, USA) is placed to suppress the lightemitted by the LED in the region overlapping with theemission fluorescent signal of PE and PerCP. The emis-sion fluorescent signal is collected by a 103 objective(NA 0.2, Lomo Optics, USA) and filtered by emission fil-ters mounted on a filter slider. Two emission filters wereused: the 595AF60 (Omega Optical, USA) for PE fluores-cence and the 695AF55 (Omega Optical, USA) for PerCPfluorescence. The filtered fluorescence is collected ontoan ST-402ME CCD camera (SBIG, USA). The recordedimages are transferred to a single board computer(665 MHz processor with 256 MB RAM, EES-3610,Evalue Technology, Taiwan) equipped with a touch-screen monitor (B084SN03 V2, AU Optronics, Taiwan).The images are analyzed using dedicated image analysisalgorithms to determine the number of CD41 T andCD81 T lymphocytes per microliter (15,20). The CD4/CD8 ratio is calculated from these data. For each test,three images from separate positions, each correspond-ing to 1.16 ll of whole blood, are recorded, and thecounts are averaged. The total volume (3.48 ll) was cho-sen to obtain a theoretical statistical Poisson variation of�5.4% at 100 cells/ll and �7.6% at 50 cells/ll (14).

The dimensions of the SP ICM instrument are 25 cm 3

25 cm 3 20 cm. The system operates on 125–240 Vor on one 12-V rechargeable battery. With a fullycharged 12-V, 7 Ah lead-acid battery, the SP ICM instru-ment can run for �2 h and analyze about 15 samples forboth CD4 and CD8 enumeration. A small 30 Ah car bat-tery is able to support the instrument for 8 h.

Ability of SP ICM on PE and PerCP detection. Toevaluate the ability of the SP ICM on PE and PerCP fluo-rescence detection, magnetic beads with the same diam-eter as cells were coated with different amounts of PEand PerCP molecules (by biotin-streptavidin conjugation)to mimic the cells with different PE or PerCP intensities.Biotinylation of paramagnetic beads (Compel Magnetic,

119HIV MONITORING BY IMAGE CYTOMETER

Cytometry Part B: Clinical Cytometry

COOH modified; 8 lm; Bangs Laboratories, USA) wasperformed by Immunicon. The biotinylated paramagneticbeads were incubated with different amounts of strepta-vidin-R-PE (SAV-PE, Caltag Laboratories, USA) or streptavi-din-PerCP (SAV-PerCP, BD, USA). To 30-ll biotinylatedparamagnetic beads, 0.25 ll, 0.3 ll, 0.4 ll, and 0.5-llSAV-PE or 3 ll, 5 ll, 5.5 ll, and 6 ll of SAV-PerCP wereadded, respectively. After 30-min incubation at RT in thedark, the PE-magnetic beads or PerCP-magnetic beadswere washed twice with PBS (0.1% BSA) under magneticseparation to remove the free SAV-PE or SAV-PerCP. The

PE-magnetic beads or PerCP-magnetic beads were resus-pended in PBS (0.1% BSA). Four groups of PE-magneticbeads and four groups of PerCP-magnetic beads with dif-ferent fluorescent intensities were obtained, and theirfluorescent GeoMean intensities (GIs) (GI 5Qn

i¼1 ai

� �1=n¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffia1a2 . . .an

np

, ai is the fluorescent inten-sity of cell i) were quantified by the FCM (FACSCalibur,BD), and their fluorescent intensity ai (ai is the fluores-cent intensity of cell i) was quantified by the SP ICM. Tocompare the fluorescent intensities obtained by the FCM(FACSCalibur using CELLQUESTTM software (BD Bioscien-

FIG. 1. Emission spectrum of the496 nm LED, excitation and emis-sion spectra of PE and PerCP, andtransmission spectra of 550-nmshort pass excitation filter, 595AF60band pass emission filter for PE fluo-rescence and 695AF55 band passemission filter for PerCP fluores-cence. Note the long PE emissiontail that penetrates through PerCPfilter.

FIG. 2. A: CD4-PE image. B: CD8-PerCP image. C: Color merge ofCD4-PE (green) and CD8-PerCP(red) images. Cells in yellow circleindicate CD4-PE labeled cells (inA), which appear dimly in the CD8-PerCP image (in B). Cells in blankcircle indicate CD81dim T cells in Band C.

120 LI ET AL.


ces)) and by the SP ICM, Log10(GI) was calculated. (FCM:

Log10ðGIÞ ¼ Log10

Qni¼1 ai

� �1=n¼ Log10ðffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffia1a2 . . . an

np Þ ¼

1n3ððLog10ða1Þþ :::þ Log10ðanÞÞ; SP ICM: Log10ðGIÞ ¼ 1

n3

ððLog10ða1Þþ :::þ Log10ðanÞÞÞ.For FCM measurements, four groups of PE-magnetic

beads were mixed with a blood sample stained withCD4-PE. The Log10(GI) of the PE-magnetic beads werecompared with those of CD41 T lymphocytes andCD41dim monocytes in the same sample. Similarly, fourgroups of PerCP-magnetic beads were mixed with ablood sample stained with CD8-PerCP. The Log10(GI) ofthe PerCP-magnetic beads was compared with those ofCD81bright and CD81dim T lymphocytes in the samesample.

In the SP ICM, the four groups of PE-magnetic beadswere respectively mixed with a blood sample stainedwith CD4-PE. CD4-PE was added to introduce the samebackground as in a real blood sample for CD4 enumera-tion. Similarly, the four groups of PerCP-magnetic beadswere mixed with a blood sample stained with CD4-PEand CD8-PerCP. CD4-PE and CD8-PerCP were added tointroduce the same background as in a real blood samplefor CD8 enumeration. In this case, CD4-PE was alsoadded because of the crosstalk of the PE emissionthrough the PerCP filter (see below).

To investigate the ability of the SP ICM on PE andPerCP detection, a known number of two groups of PEbeads or two groups of PerCP beads, which are used tomimic the CD8-PerCP-labeled CD81bright and CD81dim Tlymphocytes, were added to one blood sample. In themixture of two groups of PE beads, CD4-PE was added.In the mixture of two groups of PerCP beads, CD4-PEand CD8-PerCP were added. The images were recordedand analyzed to check whether beads with two differentintensities could be detected simultaneously in oneimage or not. The blood sample was taken from a ran-domly selected healthy donor.

PE fluorescence cross-talk in PerCP image. Figure1 illustrates the emission spectrum of the 496 nm LED,excitation and emission spectra of PE and PerCP, andtransmission spectra of 550-nm short pass excitation fil-ter, 595AF60 band pass emission filter for PE fluores-cence, and 695AF55 band pass emission filter for PerCPfluorescence. The long PE emission tail (estimated at�2%) penetrates through the PerCP filter. This cross-talkof the CD4 PE cells shows dimly in the CD8-PerCP fluo-rescence image. This is demonstrated in Figures 2A–2C.A CD4-PE image, a CD8-PerCP image, and the colormerge of these two images are shown. The CD4-PE cellslight up dimly in the PerCP image. The CD8-PerCP dimcells have approximately the same intensity as the cross-talk of the CD4-PE cells. CD8-PerCP dim cells shown inthe image are CD81dim T lymphocytes with relativelyhigh intensity, as suggested by the previous PerCP beadsexperiment. If this is the case, the SP ICM system willonly count the CD81bright T lymphocytes yielding a CD8undercount by the SP ICM when compared with theFCM.

Statistical Analysis

Based on the CD41 T, CD81 T lymphocytes counts,and CD4/CD8 ratio obtained from different methods, lin-ear regression lines were drawn, and correlation coeffi-cients (R) were calculated. Bland-Altman plots (21,22)were used to evaluate the interchange ability betweenmethods. In Bland-Altman plots, the average of the CD4or CD8 counts or CD4/CD8 ratio obtained from twomethods is plotted on the horizontal axis, and the differ-ence/average (%) is plotted on the vertical axis. Thesolid line in the plot represents the bias (the average dif-ference between the two methods), and the dashed linesin the plot illustrate the upper and lower limits of agree-ment (bias 6 1.96 SD). The 95% confidence interval (CI)of the bias is also shown.

RESULTS

SP ICM

Ability of SP ICM on PE and PerCP detection.Four groups of PE-magnetic beads and four groups ofPerCP-magnetic beads were prepared by biotin-streptavi-din conjugation, and their Log10(GI) was quantified byFCM and SP ICM. In these groups, the Log10(GI) was dif-ferent in value. We named the groups of PE-magneticbeads from the one with the lowest intensity to the onewith the highest as group PE-1 to group PE-4. Similarly,we named PerCP-magnetic beads as group PerCP-1 togroup PerCP-4.

The comparison of Log10(GI) of the PE fluorescenceof the PE-magnetic beads with those of CD41 T lympho-cytes and CD41dim monocytes is shown in Table 1.Log10(GI) values obtained by FCM and by SP ICM (5-s ex-posure time) are also given in the table. The FCM dataindicate that the Log10(GI) of CD41 T lymphocytes(2.41) is between that of the group PE-2 (2.24) and thatof the group PE-3 (2.51), and the Log10(GI)of CD41dim

monocytes (1.60) is much lower than that of the groupPE-1 (2.00). In the SP ICM, the Log10(GI) of CD41 T lym-

Table 1Comparison of Log10(GI) (GeoMean Intensity) of PE

Fluorescence of the PE-Magnetic Beads with Those of CD41 TLymphocytes and CD41dim Monocytes and Comparison of

Log10(GI) of the PerCP Fluorescence of PerCP-Magnetic Beadswith CD81bright T Lymphocytes and CD81dim T Lymphocytes

Obtained by FCM and by SP ICM (5-s Exposure Time)

FCM SP ICM

PE-1 2.00 4.30PE-2 2.24 4.55PE-3 2.51 4.84PE-4 2.56 5.00CD41 T cells 2.41 (2.30–2.60) 4.56CD41dim monocytes 1.60 (1.30–1.85) –PerCP-1 1.44 4.28PerCP-2 2.18 4.69PerCP-3 2.36 4.89PerCP-4 2.94 5.46CD81bright T cells 2.60 (2.30–2.85) 5.22CD81dim T cells 1.64 (1.18–2.30)



phocytes (4.56) is also between that of the group PE-2(4.55) and that of the group PE-3 (4.84). Therefore,CD41 T lymphocytes can be certainly detected by theSP FCM. Because of the CD3 magnetic selection,CD32CD41dim monocytes are absent in the CD4-PEimage. For a sample with a mixture of the group PE-1[Log10(GI) 5 4.30) and PE-4 (Log10(GI) 5 5.00], beadsfrom both groups are shown in the image, indicatingthat the ability of SP ICM on PE detection is at least 0.7decade.

CD81dim T lymphocytes can have a fluorescent inten-sity that is up to 50 times lower than that of the mainpopulation of the CD81bright T lymphocytes (23). Asshown in Figure 3A, in FCM (FC 500, Beckman Coulter,USA), all the cells with CD8-R-PE Texas red tandem dye(ECD) intensity above that of the large cluster of nega-tive stained cells are counted as CD81 T lymphocytes.The CD81dim T lymphocytes are those cells that fallbetween the large cluster of negative and the large clus-ter of bright positive cells.

Table 1 also shows the comparison of Log10(GI) of thePerCP fluorescence of the PerCP-magnetic beads withthose of CD81bright T lymphocytes and CD81dim T lym-phocytes. The FCM data indicate that the Log10(GI) ofCD81bright T lymphocytes (2.60) is between that of thegroup PerCP-3 (2.36) and that of the group PerCP-4(2.94), and the Log10(GI)of CD81dim T lymphocytes(1.64) is close to that of the group PerCP-1 (1.44). In theSP ICM, the Log10(GI) of CD81 T lymphocytes (alldetected CD81 T lymphocytes) (5.22) is close to that ofthe group PerCP-4 (5.46).

To investigate whether the SP ICM could simultane-ously detect CD81bright T lymphocytes and CD81dim Tlymphocytes, a known number of the beads of groupsPerCP-1 and PerCP-4 were mixed and added to a wholeblood sample to mimic the CD8-PerCP-labeled CD81dim

T lymphocytes and CD81bright T lymphocytes. In theimage obtained by the SP ICM, only the group PerCP-4 ispresent. For a sample with a mixture of the groupPerCP-2 [Log10(GI) 5 4.69] and PerCP-4 [Log10(GI) 5

5.46], beads from both groups are shown in the image.

These results demonstrate that the ability of the SP ICMfor PerCP detection is about 0.8 decade and will not beable to detect the complete range of CD81 T lympho-cytes intensities (CD81dim–CD81bright). Most likely, onlythe CD81dim T lymphocytes with a relatively high inten-sity are counted.

Comparison of SP ICM with SP FCM

The blood specimens of 145 patients were testedusing the SP ICM method and the SP FCM (tetraCXPmethod), and the obtained CD41 T and CD81 T lympho-cytes counts and CD4/CD8 ratios were compared witheach other. Figure 4 shows the linear regression plot (A)and the Bland-Altman plot (B) of CD41 T (A1, B1),CD81 T (A2, B2) lymphocytes enumeration, and CD4/CD8 ratio (A3, B3). In the FCM method, the standardautomatic CD8 gating was applied as shown in Figure3A. Excellent correlations were achieved for all the com-parisons (R 5 0.99 for CD4; R 5 0.96 for CD8; R 5

0.97 for CD4/CD8 ratio). For both CD4 and CD8 enu-meration, the SP ICM was comparable to the SP FCM(CD4: slope 5 1.06, bias 5 6.6%; CD8: slope 5 0.87,bias 5 213.7%).

The CD8 results tend to be somewhat lower than inFCM, whereas the CD4 tends to be somewhat higherthan in FCM. Consequently, the results of the SP ICM forthe CD4/CD8 are about 20% higher than in SP FCM(slope 5 1.17; bias 5 20.2%).

Counting only CD81bright T lymphocytes in FCM.We investigated whether the lower CD8 count in the SPICM is partly due to CD81dim T lymphocytes that werenot counted in the SP ICM because of its limited sensitiv-ity. The FCM data were reanalyzed, and at this time, onlyCD81bright T lymphocytes were counted. Next, theCD81bright count was compared to the CD8 countobtained by the SP ICM method.

Figure 3A illustrates the automatic gating in the FCMfor CD81 T lymphocytes counting (Scatter plot: CD8-ECD vs. CD3-PC5), in which all the cells with CD8-ECDintensity above one are counted as CD81 T lympho-

FIG. 3. Automatic gating in thescatter plot of CD8-ECD versusCD3-PC5 from a SP FCM for CD81

T lymphocytes count (A), and themanual gating for CD81bright T lym-phocytes count in the histogram ofCD8-ECD from CD31 cells (B).

122 LI ET AL.


FIG. 4. Linear regression plot (A) and Bland-Altman plot (B) of CD41 T (A1, B1), CD81 T (A2, B2) lymphocytes enumerations, and CD4/CD8 ratio(A3, B3) obtained by the SP FCM (tetraCXP) and the SP ICM. Blood specimens of 145 patients were tested. In the SP FCM method, the standardautomatic CD8 gating was applied. The error bar in each dot represents the square root of the value.



FIG. 5. Linear regression plot (A) and Bland-Altman plot (B) of CD41 T (A1, B1), CD81 T (A2, B2) lymphocytes enumerations, and CD4/CD8 ratio(A3, B3) obtained by the SP FCM (tetraCXP) and the SP ICM. Blood specimens of 96 patients were tested. In the SP FCM method, only CD81bright Tlymphocytes were gated. The error bar in each dot represents the square root of the value.

124 LI ET AL.


cytes. Figure 3B illustrates the manual gating used forthe CD81bright T lymphocytes count (Histogram: countsvs. CD8-ECD). CD3-PC5 positive cells are plotted in thehistogram (counts vs. CD8-ECD). The CD81bright T lym-phocytes were gated in the symmetric region of theCD81 histogram peak. The rest of the cells above inten-sity 1 were gated as CD81dim T lymphocytes. In ourstudy, it was found that, on average, CD81dim T lympho-cytes were about 10% of total CD81 T lymphocytes.

FCM LMD files of 96 among 145 patients were avail-able to do the manual gating for the CD81bright T lym-phocytes count. Then, only CD81bright T lymphocyteswere counted in the FCM, and the obtained CD81bright

counts and CD4/CD81bright ratios were compared withthe CD4/CD8 ratios obtained by the SP ICM. Figure5 shows the linear regression plot (A) and the Bland-Alt-man plot (B) of CD41 T (A1, B1), CD81 T (A2, B2) lym-phocytes enumeration, and CD4/CD8 ratio (A3, B3) ofblood specimens of 96 patients. The results illustratethat by the elimination of the CD81dim T lymphocytesfrom the FCM data, the slope of the CD8 countsincreased and that of CD4/CD8 ratios decreased to 1.01.Also, the biases of CD8 counts and CD4/CD8 ratiosdecreased from 213.7% to 21.9% and from 20.2% to7.1%, respectively.

DISCUSSION

The SP ICM was tested by determinations of CD41 Tand CD81 T lymphocytes counts and CD4/CD8 ratios.The system was evaluated by comparison with the SPFCM tetraCXP method on blood specimens of 145patients.

Most importantly, the CD4 values measured agreedvery well with the SP FCM data. Both the results of thecorrelation analysis and the Bland-Altman plots showedthat the SP ICM data correlate very well with the FCMdata for CD4. This also holds true for low cell concentra-tions that are important for diagnosis and treatment, thatis, below 350 and especially below 200 CD41 T lympho-cytes per microliter.

In counting CD81 T lymphocytes, a distinction has tobe made between the CD81bright cells and the CD81dim

cells. It was shown that the sensitivity of the current SPICM is such that the CD81dim cells are not, or partlynot, counted. The reasons may be the relatively highbackground of free fluorophore present in the system orthe inability of the image analysis algorithm to distinguishbetween CD81dim cells and the cross-talk of CD4-PE fluo-rescence. This may account for the �10% lower CD8counts than those obtained by the SP FCM. This couldpartly explain why the CD4/CD8 ratio is �20% highercompared to the FCM. However, the systematic errors incomparing absolute count data for different FCM instru-ments may easily reach 20% or even higher (24,25). Thiscertainly is found when completely different methods andinstruments are compared. The CD8 counts and CD4/CD8 ratios did show good linear relations with the FCMdata, and a good interchangeability of the two methodswas indicated by the Bland-Altman plots.

It should be noted that, in literature, similar differencesin enumerating CD8 cells have been reported, as, forexample, for the Guava PCA method (26), which has alimited sensitivity and gate settings compared to the con-ventional FCM that only counts CD81bright T lymphocytes.

Several options for further improvement may beimplemented in the future. An obvious way to preventthe interference of the cross-talked CD4-PE cells onCD81dim T lymphocytes enumeration is to perform CD4and CD8 enumeration in two separate analysis chambers.However, this will increase the cost per test and doublethe sample preparation steps. The limited sensitivity ofthe SP ICM system in the detection of low intensity fluo-rescence is due to the presence of free fluorophore andferrofluid in the sample. The resulting high backgroundcompresses the dynamic range of the system to a muchhigher degree than in FCM where no ferrofluid is presentand the free fluorophores are diluted. In the current SPICM system, a sample chamber with inner depth of 4 mmwas used. It is expected that by using a thinner chamber,the background could be strongly reduced, which maylead to better detection of CD81dim T lymphocytes.

Our portable, battery-operated, and computer-con-trolled automated instrument is low-cost (componentscost: US$3,000; assay cost: US$3 for CD4 and CD8), yetreliable, and robust. No alignment or calibration isneeded. The immunolabeling method and the instrumentare easy-to-use even for less-trained operators. In conclu-sion, our system is a good candidate for HIV monitoringin point-of-care settings of resource-constrained coun-tries. In addition, a great application of this CD4/CD8 ra-tio determination system is to establish a presumptive di-agnosis of HIV infection in HIV-1 exposed infants underthe age of 18 months in resource-constrained countrieswhere PCR or RT-PCR methods are not available (27,28).A larger group of HIV1 patients will need to be tested inresource-constrained settings to determine whether the20% undercount of CD4/CD8 ratio is consistent or not.If so, the 20% can be used to correct the CD4/CD8 ratioundercount before the instrument is improved to solvethe CD8 undercount.

ACKNOWLEDGMENTS

We thank I. Vermes, M.D. Ph.D. and C.H.H. ten Napel,M.D. Ph.D. from MST hospital, Enschede, for kindly sup-plying fresh blood samples and FCM data. We acknowl-edge all patients and healthy donors whose blood wastested in this study.

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CD4 and CD8 enumeration for HIV monitoring in resource-constrained settings