Emerging Microscale Technologies for Global Health: CD4+ Counts - Bionanotechnology: Global Prospects - page 131

requirement. for. laboratory. space,. reliable. power. supply,. and. refrigerated. storage.. To. be.
more.suitable.for.rural.areas,.two.manual.CD4 + .cell.counting.methods.have.been.devel-
oped:.Dynabeads.and.Cytospheres.(Table.6.1)..However,.they.are.still.considered.expen-
sive,.labor-intensive,.and.low.throughput.
Dynabeads. is. a. manual. CD4 + . cell. counting. method. that. relies. on. the. use. of. MAb-
coated. magnetic. microbeads. 35 . Three. steps. are. involved:. depletion. of. monocytes. from.
whole. blood,. isolation. of. CD4 + . cells,. and. staining/counting. nuclei. in. a. hemocytometer..
In. the. irst. step,. CD14. MAb-coated. Dynabeads. are. added. to. fresh. EDTA-anticoagulated.
blood..CD14 + .MAb.speciically.binds.to.monocytes,.which.also.express.CD4 + .on.the.cell.
surface.. When. applying. a. magnetic. microbead. concentrator,. the. monocyte-CD14. MAb-
Dynabead.complex.is.separated..The.supernatant.is.transferred.to.a.new.tube.containing.
Dynabeads. coated. with. anti-CD4 + . MAb.. After. the. CD4 + . cell. separation,. CD4 + . cells. are.
lysed.and.the.nuclei.are.stained.with.acridine.orange.and.counted.under.a.luorescence.
microscope.. In. a. modiied. format,. the. number. of. free. CD4. Dynabeads. decreases,. with.
less.Dynabead.input.and.more.washing,.enabling.a.reliable.CD4 + .cell.count.under.a.light.
microscope. 36 .Dynabeads.CD4.only.requires.a.light.microscope,.a.hemocytometer,.and.a.
counter,.signiicantly.reducing.the.initial.setup.cost,.and.only.costs.approximately.$5.per.
assay..Once.CD4 + .cells.are.lysed,.however,.counting.needs.to.be.performed.within.1.hour,.
which.restricts.the.batch.size.to.less.than.six..Therefore,.this.manual.method.is.suitable.for.
local.clinics.where.throughput.is.not.a.concern..In.a.resource-limited.context,.Dynabeads.
CD4. showed. a. high. correlation. of. 0.89. with. low. cytometry.. However,. Dynabeads. CD4.
underestimated.the.CD4 + .cell.count.up.to.269.cells.when.the.CD4 + .cell.count.was.above.
1,000.cells/µl. 35 .This.was.further.conirmed.in.a.recent.study,.in.which.the.median.differ-
ences.between.Dynabeads.and.low.cytometry.were.1,.16,.24,.and.90.cells/µl.with.CD4 + .cell.
counts.of.<200,.200-350,.350-500,.and.>500,.respectively. 37
The.Cytosphere.assay.is.another.bead-based.manual.CD4 + .cell.counting.method. 38 .It.
uses.CD4 +. MAb.(MY4)-conjugated.latex.spheres.to.enumerate.CD4 + .T.lymphocytes.under.
a.light.microscope..MY4,.however,.cannot.differentiate.the.CD4 + .molecules.between.CD4 + .
T.lymphocytes.and.monocytes..To.reduce.the.overestimation.caused.by.monocytes,.the.
Cytosphere.assay.uses.anti-CD14 + .monoclonal.antibody.to.block.monocytes..The.blocked.
monocytes.form.a.golden.coloration,.which.is.easily.distinguished.from.CD4 + .cells..The.
following. counting. is. carried. out. on. a. hemocytometer,. on. which. cells. attached. with.
three.or.more.latex.spheres.are.recognized.as.CD4 + .T.lymphocytes..Evaluation.in.India.
showed. that. this. FDA-approved. assay. had. high. correlation. with. low. cytometry. (R. =.
0.97),.and.it.was.reliable.in.identifying.AIDS.patients.with.CD4 + .cell.counts.below.200.
cells/µl. 38 .However,.a.recent.large-scale.study.demonstrated.that.the.Cytosphere.assay.
underestimated.CD4 + .cells.up.to.210.cells/µl.when.it.is.above.500.cell/µl,.with.a.correla-
tion.coeficient.of.0.78. 37
Based. on. the. principle. of. Cytospheres,. a. new. microbead-based. assay. was. developed,.
utilizing.a.different.anti-CD4 + .monoclonal.antibody.(MT4). 39 .This.antibody.has.a.stronger.
afinity.to.CD4 + .T.lymphocytes.than.monocytes,.eliminating.the.need.for.monocyte.block-
ing.by.anti-CD14 + .MAb,.as.in.the.Cytosphere.assay..In.this.assay,.MT4.MAb.is.coated.on.
latex.microbeads.and.used.to.capture.CD4 + .cells.from.whole.blood..Once.bound.to.CD4 + .
cells,.latex.microbeads.and.CD4 + .cells.form.a.rosette.structure,.which.can.be.easily.identi-
ied.under.a.light.microscope..On.a.hemocytometer,.cells.with.three.or.more.microbeads.
are.recognized.as.CD4 + .cells.and.manually.counted..The.evaluation.compared.with.stan-
dard.low.cytometry.showed.a.high.correlation,.with.R 2 .=.0.941.among.60.health.people.
and.140.HIV-1.infected.individuals..In.another.strategy,.this.MT4.MAb.is.used.to.deplete.
CD4 + . cells. from. whole. blood. to. facilitate. CD4 + . cell. counting. 40 . With. this. method,. white.