Emerging Microscale Technologies for Global Health: CD4+ Counts - Bionanotechnology: Global Prospects - page 130

be.set.up.in.centralized.laboratories.due.to.the.need.for.laboratory.infrastructure,.such.as.
constant.power.supply.and.skillful.operators.
EasyCD4.is.a.microcapillary.cytometer.that.relies.on.the.use.of.a.microcapillary.to.main-
tain.single-cell.suspension. 27 .This.design.offers.two.advantages..First,.the.use.of.a..capil-
lary.excludes.the.need.for.sheath.low,.and.thus.reduces.the.consumption.of.iltered.water..
This.is.particularly.important.since.clean.water.is.not.readily.available.in.most.resource-
limited.settings..Second,.a.small.volume.of.blood.(10.µl).is.needed.for.CD4 + .cell.counting..
To.achieve.speciic.CD4 + .detection,.EasyCD4.utilizes.two-color.conjugated.anti-CD3 + .and.
anti-CD4 + .MAbs,.which.are.conjugated.with.phycoerythrin.(PE)-Cy5.and.PE,.respectively..
Since.anti-CD3 + .MAb.targets.an.epitope.on.the.T.cell.receptor.(TCR),.CD3 + .gating.excludes.
the.interference.of.monocytes.(CD3 - ),.which.also.express.CD4 + ..These.two.MAbs.are.irst.
added.to.10.µl.of.EDTA-containing.whole.blood.and.mixed.gently..Red.blood.cells.in.the.
mixture.are.then.lysed.prior.to.CD4 + .counting..Evaluation.studies.have.demonstrated.that.
EasyCD4.has.a.strong.correlation.with.FACSCount.(R 2. =.0.92.~.0.99).and.can.be.established.
with.quality.assurance..Most.importantly,.EasyCD4.can.accurately.identify.AIDS.patients.
with. CD4 + . cells. below. 200. copies/µl. 20,27,28 . However,. a. recent. study. shows. that. EasyCD4.
gives.a.consistently.higher.CD4 + .cell.count.(25.cells/µl).than.FACSCount. 29
CyFlow.(Partec).is.a.single-platform.volumetric.low.cytometry. 21 .To.reduce.cost,.it.uses.
only. anti-CD4 + . MAb. conjugated. with. PE,. and. CD4 + . cells. are. counted. by. a. single. green.
solid-state.laser.(532.nm)..The.simple.testing.protocol.involves.no.lyse.and.no.wash,.reduc-
ing.the.hands-on.time.and.systematic.error..In.brief,.100.µl.of.whole.blood.is.mixed.with.
10.µl.of.anti-CD4 +. MAb.and.incubated.in.darkness.for.10-15.minutes,.where.it.is.then.fol-
lowed. by. the. addition. of. 800. µl. of. no-lyse. buffer.. For. the. volumetric. measurement,. two.
electrodes. are. immersed. into. the. mixture. at. different. levels.. The. actual. counting. takes.
place.when.the.luid.level.falls.between.these.two.electrodes.during.aspiration..Although.
CyFlow. involves. no. complex. gating. strategies. or. any. optical. alignment,. it. still. requires.
substantial.technical.expertise.to.differentiate.between.monocytes.and.CD4 + .T.lympho-
cytes,.since.they.both.express.CD4 + .molecules..Clinical.evaluations.have.been.performed.
in. Thailand, 21 . Zimbabwe, 30 . and. Malawi, 31 . showing. high. correlations. with. FACSCount.
(R 2 =.0.92.to.0.99).
Pan-leucogating.(PLG).is.a.dual-platform.low.cytometer..Compared.to.conventional.four-
color.low.cytometry,.it.utilizes.only.two.MAbs,.anti-CD45 + .and.anti-CD4 + ,.for.CD4 + .cell.
count.to.reduce.cost..CD45 + .is.a.pan-leukocyte.marker,.and.it.is.used.to.identify.white.blood.
cells..After.gating.with.anti-CD45 + ,.white.blood.cells.are.further.gated.with.anti-CD4 + .to.
determine.the.percentage.of.CD4 + .cells.in.white.blood.cells.(CD4%)..CD4 + .cell.count.is.then.
calculated.by.multiplying.CD4%.with.white.blood.cell.count,.which.is.obtained.from.a.hemo-
cytometer..The.advantage.of.this.approach.is.that.PLG.uses.less.variable.white.blood.cells.
(CD45 + .population),.instead.of.total.T.lymphocytes,.in.the.gating.strategy,.which.increases.
reproducibility.and.enables.testing.day-old.samples. 32 .Evaluation.studies.in.Thailand.and.
Barbados.have.shown.that.PLG.is.highly.correlated.with.standard.low.cytometry,.with.an.
R 2 .of.0.95.for.absolute.CD4 + .cell.count. 22,33 .PLG.can.also.be.adapted.to.single-platform.low.
cytometry,.in.which.bead.count.rate.(BCR).is.introduced.for.proactive.quality.control. 34 .This.
simpler.low.cytometer.proves.to.be.reliable,.inexpensive,.and.has.high.throughput.capabil-
ity,.showing.the.promise.of.guiding.ART.in.resource-limited.settings.
6.2.3 Manual CD4 + Cell Counting Methods in Resource-Limited Settings
Although. modiied. low. cytometry. techniques. reduce. the. initial. cost. for. instrument.
setup.and.reagent.cost,.they.can.only.be.established.in.centralized.laboratories.due.to.the.