Download Detection of HIV-1-Infected Cells From Patients

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Detection
of HIV-1-Infected
Using
By Robert
We
have
demonstrated
hybridization
infected
Our
cells
assay
DNA
from
(HIV)-infected
assay
and
rare,
were
PBL.
The
same
past
It
the
HIV
infected
the
production
of
of
suggest
that
with
of
blood
to the
ofacquiring
remains
in
blood
donors
being
in the
in situ
genome
directly
situations
of
all
would
antibody
would
be helpful
situ
immunlogic
whether
lose
method
in assessing
and in monitoring
In
who
following
and quantitative
tive
antiviral
hybridization
methods,
since
proteins
Blood, Vol 74, No 6 (November
of HIV
infection.
and convenient
to detect
would
increase
fail
the
(eg,
the
ability
acute
to
infection.7
to the
in infants
acquired
in
Finally,
HIV-infected
the
history
therapy
or vaccine
distinct
it is able
to detect
pp 2295-230
cells
of infection
development.
advantages
are expressed.
1), 1989:
those
a sensi-
ofdetecting
natural
infected
Such
& Stratton,
In situ
without
and
speed,
in three
sensitivity,
detection
and
indicates
clinical
that
research
and
Inc.
in
report
cells
hybridization
of
cells in frozen
Harper
et al9
demonstrating
in the
lymph
patients
to any other
situ
viral
time
in
probe.
situ
HIV-infected
peripheral
complex
shown that
of culture
In these
technique.
methodology”
labeled
(355)
nonisotopic
and
and AIDS-related
Bush et al’#{176}
have
at an earlier
detection
hybridization
cally
the detection
nodes
can be used to detect virus-positive
peripheral
blood
mononuclear
cells
cocultures
employed
This
compared
reports
the in
a radioisotopi-
report
improves
(ISH)
to the
hybridization
cells
from
and applies
detection
of
cells.
METHODS
Preparation
of
Blood
ture.
was
peripheral-blood
obtained
lymphocytes
by venous
puncture
and
cocul-
the lymphocytes
and
separated
by Ficoll-Hypaque
(FH)
centrifugation.
Peripheral
blood
mononuclear
cells were isolated
from seronegative
and seropositive
individuals
(3 x 106 cells) and cocultured
with cells (3 x 106) from
healthy
with
seronegative
donors
Research
Triangle
Park,
HEPES-buffered
RPMI
had been stimulated
1640
2 zg/mL
(FCS),
serum
that
for 3 to 4 days
(PHA) (2 .tg/mL, Wellcome
Diagnostics,
NC). Cocultured
cells were maintained
in
phytohemagglutinin
From
the
Genetics
supplemented
polybrene
Departments
and
School,
cells
of
with
(Sigma
Worcester.
20%
fetal
Diagnostics,
calf
St Louis,
March
by a contractfrom
1, 1989;
Health
(NIH)HB-67027
18066
to
R.H.S.
Ave N,
Lake
and
reprint
J.B.L..
Worcester,
indicate
© 1989
Molecular
Medical
This
5, 1989.
to Robert
and
to
Heart
article
Institutes
NIH
J.L.S.
of
grants
HD
J.L.S.
is
an
Association.
H. Singer.
of Massachusetts
MA
in accordance
National
J.L.S.;
42257
American
costs ofthis
payment.
and
ilL
ofthe
requests
July
the NHLBI.
University
The publication
charge
accepted
to R.H.S.
Investigator
Address
Pediatrics,
of Massachusetts
MA.
Supported
of Cell Biology.
Biology,
University
Submitted
established
Cell
Microbiology.
“advertisement”
over
hybridiza1
positive.
viral
likeli-
an appropriate
maintain
HIV-
to mount
capture
directly,
hemophiliacs
for
patient
antigen
nonrathe
prior
informative
offers
or not viral
While
where
passively
the diagnosis5
and
obscure
or
sero-
infected
that continual
result
in more
individuals
not
mothers
response6
specific
are
individuals
and
the
immune
response.
A method
to
also be extremely
useful in those
antibodies
antibodies
l-IIV-infected
antibody
cells
of a virus-specific
directly
the
transfusion
assay
HIV-infected
HIV-seropositive
maternal
sensitive,
in infected
when
period”
hybridization
of detecting
generation
detect
HIV
“window
of a rapid,
development
dioisotopic
from
antibody.
it is probable
community
will
the
as virus
The
tool
a further
symptomatic
from
products.
blood
hybridization
in the
HIV
of HIV
through
(34%)
a
situ
cells
of individuals
with AIDS
by in situ hybridization.
in situ
of Ward
et a14
are infected
are obtained
infection
(1
the
occur
to be acutely
appearance
HIV
I
of cells,
blood
products
in 40,000),
heterosexual
low
spread
may
and
HIV-contaminated
who are thought
prior
host
The recent
studies
as 460 individuals/year
donors
viremic
hood
the
HIV-infected
risk
The
by
many
blood
(ARC)
in a variety
as
by Grune
published
been
enters
fourth
positive
infants.
useful
HIV-infected
have
secretions
HIV
than
of 35
always
capture.
hemophilia
was
have
monocytes/macrophages
Viral replication
precedes
lymphocytes.3
because
negative
Once
be
brain
of
of HIV
In
whereas
one
ISH
antigen
first used to detect
HIV-infected
tissue obtained
from AIDS
patients.8
tion
been
vaginal
coculture.
positive.
(37%)
that
as
HIV-infected
of ISH and nonisotopic
will
a 1989
available
regarding
HIV
infection
in
ofspread
less
found
diagnosis.
has
type
of
detected
eight
was
samples
of our
in 1 2 out
of
it
normal
virus
replicates
tissue
antibody
as
HIV
These
and
and
it
times
as virus
confidence
of approximately
and
individuals.
symptoms.
absence
semen,
the sources
infects
blood
(CD4)
I-helper
the
blood,
out
a Dupont
Americans
of
coculture,
(AIDS)
has
United
States
seroprevalence,
is currently
quantitation
represent
virus
including
and
that
and
rate
a
It
positive
half
detected
a
in-
using
shorter
hybridization
from
with
immunodeficiency
to noninfected
host,
coculture
to I .5 million
Little
information
detection
and
assay
within
derived
investigated
of
using
L. Sullivan
kit.
same
over
population
HIV-seropositive
after
a mortality
human
is known
harbor
with
surveys
1 million
with
(HIV
l).2
the direct
vivo.
on
that
infected
and
and
were
10 years
Based
estimated
samples
in
virus
identify
immunodeficiency
syndrome
over 60,000
individuals
in the
affected
the
Patient
before
samples
be performed
unambiguously
hemophiliacs
CQUIRED
60%.’
it can
to
and John
the
detected
positive
Patients
antigen-capture
often
HIV
(PBL)
identify
B. Lawrence,
detected
individuals.
immunodeficiency
From
Hybridization
P24
HIV-
a biotinated
to
Jeanne
in situ
detect
lymphocytes
in that
cell.
analyzed
to
of
human
enough
HIV-seropositive
fants
S. Byron,
asymptomatic
blood
positive
used
phosphatase
is rapid
is sensitive
single,
over
to
alkaline
Kevin
In Situ
nonisotopic
be
detection
peripheral
and
This
day
can
the
hybridized
streptavidin
H. Singer,
seropositive,
on
Nonisotopic
a sensitive.
assay
is based
probe
cells.
that
(ISH)
Cells
PhD.
Department
Medical
School,
55
01655.
article
must
with
were defrayed
therefore
18 U.S.C.
be
section
in part
hereby
by page
marked
1 734 solely
this fact.
by Grune
& Stratton,
Inc.
0006-4971/89/7406-0060$3.00/0
2295
to
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
SINGER
2296
MO), 10% interleukin-2
(IL-2, Cellular
and 12.5 g/mL
gentamicin
(GIBCO,
Products
Inc. Buffalo,
Grand
Island, NY)
weeks.
and were
Cultures
fresh
were
split
twice
weekly
supplemented
with
normal mononuclear
cells at 7-day intervals.
Immunofluorescence.
Cells were mixed with a 1: 100 dilution
of
patient
serum (from a HIV-seropositive
individual)
in phosphatebuffered saline (PBS) and, after a series of washes in PBS, were
detected with fluorescein-conjugated
rabbit antihuman
IgG (Cappel
Laboratories,
Organon
Teknika,
West Chester,
PA). Cells were
visualized
under epifluorescence
optics
at 40 X objective.
Dot blot.
Cellular
pellets (2 x 106 cells) were washed and
three
times
with
phenol
chloroform
after
sodium
LYMPHOCYTES
ON SLIDE
dodecyl
sulfate
(SDS)
(1%) treatment
of the cells. Isolated
nucleic acids
were purified by ethanol precipitation,
resuspended in high salt (0.3
mol/L
Na acetate), and blotted onto nitrocellulose
filters. Filters
were
hybridized
with
32P-labeled
HIV
genomic
probe’2
BIOTINATED
DNA
PROBE
CELLS
(3 x 108
overnight,
washed,
and
exposed
for 72 hours to radiographic film with intensifying
screens.
Probe preparation
by nick translation.
An amount
of 1.5 ML of
400 mol/L
biotin dUTP’3
(BRL,
Gaithersburg,
MD) or the
corresponding
dATG mix for radioactive
probes
(or for radioactive
probes, 355-dCTP,
Amersham
1,000 Ci/mmol/L,
diluted
1:3 with
unlabeled
dCTP and used at a final concentration
of 6 imol/L),
I
tL of dACG
mix (dATP,
dCTP,
dGTP
600 mol/L
each, PL
Biochemicals,
St Louis, MO), or dAGT mix for radioactive
probes, 1
sL of lOX nick translation
buffer (0.5 mol/L Tris Cl, pH 7.2; 0.1
mmol/L
Mg504;
I mmol/L
dithiothreitol),
500 sg/mL
bovine
serum albumin (BSA, Pentax Frac V) 5-sL sterile glass distilled
1 &Lof0.l
H20;
g/tL
HIV
DNA
(pJM
[HIV-118.9[121
in
SSC
PUT
ONTO
HYBRIDIZATION
Situ
PROBE
TO
HYBRIDIZES
VIRAL
ACIDS
NUCLEIC
IN
CELL
contain-
of HIV-1
DNA),
I L of DNase
(final 34 ng/mL:
concentration
determines
the probe size; large probe sizes cause
background),
I zL of DNA Polymerase
I (Boehringer).
Incubation
is 3 hours at I 5#{176}C,
then 90 zL of 50 mmol/L
EDTA
and I L of 10%
SDS is added. Purification
from incorporated
nucleotides
is with a
sterile
G50 sephadex
“spin”
column
packed in a 1 mL disposable
syringe.
In situ hybridization.
The schematic
in Fig I outlines
the
following
methodology.
Cells
were washed
in PBS, and 25 x iO
cells in 25 zL were applied to each well of a multiwell
serologic slide
(Celline, 5-mm wells). The excess fluid was immediately
withdrawn
by the pipette, leaving
cells to be air dried (10 minutes),
fixed in 4%
paraformaldehyde
in PBS (5 minutes), and stored in 70% ethanol at
4#{176}C.
From 1% to 4% (25,000
to 100,000; median 50,000) of the cells
applied actually remain adhered
to the slide. At appropriate
times,
slides stored in 70% alcohol are rehydrated
in a 10-minute
PBS, 5
mmol/L
MgCI bath, then 10 minutes in 0.5% triton/0.5%
saponin
solution,
washed in 2X SSC, treated
for 10 minutes
in 0.1 mol/L
triethanolamine
and 0.25% acetic anhydride,
washed again in 2X
SSC, and incubated
for 2 minutes at 70#{176}C
in 70% formamide
in 2X
8.9
VIRAL
COMPLEMENTARY
cpm/tg)
ing
AL
NY),
for 3
PHA-stimulated
extracted
ET
kb
before
plunging
into
70%
ethanol
and
dehydrating
through
AVIDIN
BINDS
ENZYME
COLOR
Fig
1
detection
shorter
convenient)
protocol,
the signal
is diminished.
Forty
nano-
Schematic
protocol.
.
graded ethanol and air drying. This makes viral DNA as well as
RNA accessible
for hybridization.
While the cells can be hybridized
directly
out of the 70% ethanol
after rehydration
in PBS, for a
pretreatment
CONJUGATED
ALKALINE
of the
in a humidified
-
TO
PHOSPHATASE
TO
BIOTIN
ON
PROBE
DEPENDENT
GENERATING
nonisotopic
SYSTEM
in situ
37#{176}C
incubator.
Slides
hybridization
and
are rinsed
in 50%
grams of probe DNA is used, which can be obtained
by aliquoting
40
iL of nick-translated
probe into a microfuge
tube and lyophilizing
it
with 4 tL of carrier
nucleic acids (10 mg/mL
of sheared
salmon
sperm and tRNA).
Five microliters
ofdeionized
formamide
is mixed
with the lypholite and placed in a 90#{176}C
heating block for 10 minutes.
Hybridization
buffer
is prepared
by mixing
30 pL 20X SSC; 30 ML
BSA, 60 ILL dextran sulfate (50% solution, autoclaved
in water) and
for 30 minutes at 37#{176}C,
then placed in 2X
SSC for 30 minutes and finally in 1X SSC for 30 minutes.
Probes obtained from Molecular
Biosystems, Inc (Dupont SNAP
probe, San Diego, CA) were used at a concentration
of 1 ng per
hybridization
(6 nmol/L
concentration).
Cells were hybridized
in
5x SSC, 0.5% SDS, and 1% BSA at 50#{176}C
for 20 minutes
in a
30 ML H2O. Slides are then quickly used with the deposition
of 5 ML
of the heated probe mixed rapidly with an equal amount of hybridization
buffer onto each serologic
well (10 ML total), covered with a
small strip of parafilm,
and placed for 3 hours (or overnight
as is
minutes
formamide
in 2X
humidified
detection
SSC
environment.
in
I X SSC
Samples
at room
of the alkaline
Detection
ofbiotinated
by biotinated
alkaline
were
then
temperature
phosphatase
probes.
phosphatase
for
washed
at 42#{176}C
for
5 minutes
streptavidin
after the method
5
The
below.
was as described
Originally
each.
ofSinger
followed
et al’4
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DETECTION
Table
OF HIV- 1 -INFECTED
1 . Detection
Seronegative
of HIV-Infected
Individuals:
2297
CELLS
Cells
Comparison
With
In Situ
From
Seropositive
and
of Antigen-Capture
was
ELISA
In Situ
Patient
Population
(N)
Capture
(%)
Hybridization
Positive (%)
in Table
exposed
to the
SSC
Coculture
Seronegative
hemophiliacs
(2)
Seropositive
hemophiliacs
(34)
Seronegative
normal
Seropositive
0
controls
infants
(8)
(1 5)
for
18 (54%)
0
0
5 (33%)
8 (53%)
color
normal
controls
hemophiliacs
Seropositive
infants
‘At
four
sample
hybridization,
samples
was
tested,
greater
clear
over
and
cells
put
on each patient
For
the
P24
directly
For
on
were
by direct
slides
tested
was
tested,
and
days was positive.
the
For
antigen
capture
200
pg/mL
detection,
and
of p24
the
antigen
patient
processed
for the presence
ELISA,
z1 of supernatant
as
of p24
mononudescribed
antigen
detection.
infants
with detectable
serum p24 antigen
had symptomatic
one each with AIDS and progressive
generalized
lymphade-
HIV infection,
nopathy.
detection
or
the
were taken
detection
aliquots
of the same cells.
mined
dilution.
by
calculation
Since
the
at
background
by
After
Nacl,
results
in 4X
MgCI2)
(BRL,
Gaithers-
phosphate
developed
standard
washing
50 mmol/L
tetrazolium
20
to
30
autoradiographic
AND
(BRL,
minutes).
technique;
DISCUSSION
of HIV
in a variety
of in situ hybriddetection’’6
to
of cellular
samples.
ments
by
contributed
used the producer
a majority
of cells
negative
and yet permit
infected
cell with
cells.
lines of HIV
are highly
positive
(CEM
A
treatments
prior
a
no
progressive
for rapid in
Initial
experior H9)
to optimize
For instance,
conditions
that
of cells on slides were monitored
of the cells,
Since
et al,9 it was apparent
that the applicato HIV
detection
in clinical
samples
approach
to the improvement
of methodologies
situ hybridization
and detection
was used.
in which
protocol
provided
optimal
by microscopy.
to probe
application,
7
-
8
0
9
I
2
I0
3
II
4
5
each
6
undiluted
sample
contained
7% positive
cells. as determined
by in situ
hybridization.
this provided
the
numerator
in the serial
dilutions. (B) Samples
in (A) taken
for dot-blot
analysis.
mol/L
required
that it be sensitive,
convenient,
signal to be evident
in an extremely
rare
for micros-
(A) Dilution
curve
(0-0):
percent infected
cells (average
of
10 fields).
(0-0)
Identical sampies measured
by immunofluorescence.
(X-X)
The expected
percent
of infected
cells deter-
clinical
the cells were put into a pH
bromochloroindolyl
of serially
copy.
Methods
of detection
used an indirect
immufluorescence assay and in situ hybridization.
In addition.
dot blots
were
made from RNA isolated
from
0.1
dilution;
the report of Harper
tion of the approach
diluted
infected
cells: in situ
hybridization
versus dot blot or
immunofluorescence.
CEM
cells chronically
infected
with
HIV were
serially
diluted
1:1
with uninfected
CEM cells. and
aliquots
each),
nitroblue
and
was
uninf
Detection
for 30 minutes.
In this work we describe
the application
ization
followed
by enzymatic,
nonisotopic
Fixation
2.
3. The
was for 3 to 5 days.
conditions.
retention
Fig
Tris,
with
RESULTS
3 (37%)
cells) was sufficient
to 40
direct
mol/L
1 2 (34%)
50,000
by Dupont:
corresponding
positive.
samples
studied
culture
if any one of those
as described
readings
were
the
cell per well (average
considered
Serum
tBoth
positive
1
assayed
were
previously.
2 1 dayS,
2 and
0
4 ( 1 1 %)
2 (25%)t
determination.
were
0
(8)
intervals
a positive
for
(7)
(35)
was considered
in situ
(0.1
development
Seropositive
in Figs
cells
burg, MD; 1:230 dilution)
Gaithersburg,
MD;1:300
detection’
Seronegative
hybridized
development
Isotopic
Direct
presented
washes for 10 minutes
(three
9.5 solution
0
8 (24%)
data
for the
I were obtained using streptavidin-alkaline
phosphatase conjugate
(Dakopatts,
Santa Barbara, CA), which removes
a step in the detection
protocol and improves
the background.
The
conjugate
is used after dilution 1:250 into 4X SSC with 1% BSA and
Hybridization
p24 Antigen
Positive
used
reported
A
Dilution (ha)
B
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2298
SINGER
amounts
cell
of viral
being
a
uninfected
seen
w
I-
single
8ad
may
cells.
4
C.)
88.)
Ui
0.
a.
in 50,000
On
negative
Fig 3.
Kinetics
infected
AFTER
of infection
cells
by
equivalent
sensitivity
of indirect
magnification
of normal
the higher
tendency
in situ
T lymphocytes:
hybridization
of antigen
Deteccapture.
and hybridization
conditions,
penetration”
and accessibility
monitored
followed
by isotopic
by scintillation
ly. Finally
nonisotopic
in chromogen
development,
such
detection
counting
influence
molecules,
probe
were
using
32p-labeled
probe
or detection
nonisotopical-
quantitative
data
were
obtained
the positive
cells under the microscope
as a percent
of total cells. Once the
of
HIV
with
as RNA
dot
possible.
infected
per
1,000.
Another
follow
where
blot’7
and
antigen
we have defined
capture
by
and expressprotocols
for
methods’8
sensitivity
We evaluated
this
in which
positive
one in i05 cells).
was
as the ability
form of sensitivity
cells were diluted
Virus-positive
Hence
the
per
cell
cells
were
HIV
never
that
level
With
and
a
to score
a
samples
are
of positive
cells
to
by a
(to
serially
cell
expressed
linked
3).
overnight
using
tested
a
(kindly
provided
mixture
the
that
in the
initial
these
positive
topic
in situ
culture
cell
cells
could
hybridization
(Fig
2A).
were
when
The
positive
be accurately
diluted
false-positive
(I
in 14 cells),
and
detected
by noniso-
time
required
the positive
tional
to the
to one infected
rate
was
cell
determined
on an uninfected
population
ofcells
and was found to be zero
(no false positives
seen in 100,000
cells).
Because
of the
visual
requirements
for characterizing
a cell as positive
(the
be
completely
colored,
indicating
significant
as in
many
ELISA
assay.
to approximately
limit
approximately
mixture
We
used
40
level
of p24
I cell
after
MA)
color
and
found
development
approximately
was
used
to
proporon the
same
to be the less
levels
were
from
(about
antigen
would
per
2500
1 1000
positive
correspond
negative
would
(this
are
capture
as a confident
expression
pg/mL)
5
determined
Fig 3 to determine
by
pg/mL
per
an
recently
by in situ hybridization)
cell
positive
have
oligonucleotides
determination
(10
and only
methods
(defined
I positive
of comparable
of background
both
This
of detection
were
It was found
it is possible
positive
per
lines.
complexity.
test
as a result
cells
cell
We
sufficient
from
producer
Billerica,
hybridization.
results
a
determination.
enzyme-linked
DuPont,
ELISA
samples,
for
lower
situ
positive
required
20
in the
directly
of HIV
probes,
was decreased
capture
detected
oligonucleo-
of three different
enzymeon the average,
25% of the
chromagen.
for
cells
increase
antigen
on equivalent
how
of
In situ
probes
of in situ hybrids
probe
the
by MBI
detect
The
to
DE).
labeled
copies
detection
for
antigen
probes
with
HIV
1 week
phosphatase
compared
mixture
detected,
Normal
p24
and
fewer
nick-translated
exposure
Since
cells
The
oligonucleotides
detected
alkaline
where
in a
acid
with
for
biotinylated
genomic
cells
or
phosphatase,
and nonisotopic
(Fig
sensitivity
intervals
to as
cell.’
nucleic
cells.
(Dupont,
Wilmington,
using
isotopically
to
cell
is to
per
infected
hybridization
alkaline
nick-translated
RNA
in infected
to
the amount
in viral
were
situ
when
due
and the
positive.
of detectable
at daily
in a system
the isotopic
cells
number
increase
assay
done
of
eventually
of HIV
the increase
conjugated
method
7% of the
in
and
probes)
limited
where
is increasing,
by autoradiography,
probes
limit
sensitivity
time
with
lymphocytes
by
(SNAP
the
of cellular
DNA
900 (Fig 2B). The
was
of copies
as an
streptavidin
the
that
comparative
per cell
in the
I
pg/mL).
Approximately
assess
B) and sampled
are
found
cells by immunofluorescence
to only about one infected
represent
capture
by ELISA
hybridization
was
Both
was
required
by epifluorescence
weakly
fluorescent
cells as false
increase
as well
detected
of our samples.
it
of a culture
detection
using
in many
to
should
population
sensitive
immunofluorescence.
must
mean
a low
immunofluorescence
acids
indirect
cell
not
since
of thousands
samples
14,000
does
of infected
in our hands
nucleic
as tens
diluted
by uninfected
cells and the percent
positive
cells at
each dilution
determined
by in situ hybridization.
Aliquots
were also collected
for dot-blot
filter
hybridization
or the
per
This
is seen,
infection
many
tide
were established,
comparison
of in situ
conventional
methods
of virus detection
the rare cell.
experiment
less than
signal
model
the
of viral
by
By providing
information
as to numbers
of cells
in situ, hybridization
provides
a means
to evaluate
sensitivity,
detect
mixing
all of which
to the target
detection
was evaluated
by variation
development
time or conditions
of streptaviphosphatase
incubation
and
washing.
After
din-alkaline
detection
hybridization
sufficient
to identify
Hence,
detection
was also accurate
(HTLV-III
them
represents
dot-blot
filter
hybridization
to one infected
cell per
of
PHA-activated
ing
is significant,
samples
INFEC11ON
Normal
T lymphocytes
were cultured
in vitro for 3 days with IL-2
and PHA and than were infected
with HIV-1 at low multiplicity
of
infection
(MOl). At daily intervals.
including
just after exposure
to
the virus (day 0). cell samples
were taken for in situ hybridization.
Three
methods
of in situ hybridization
were
used:
isotopically
labeled
probe (open squares).
nonisotopically
labeled
probe (open
circles),
and conjugated
oligonucleotides
(-).
In addition.
p24
was measured
in the supernatant
of the culture
using an ELISA
method
(open
triangles)
(Dupont
first-generation
kit). Control.
uninfected
cells were
processed
at days 0. 1 . and 3 for the ELISA
assay
for p24 (x---x).
For the in situ hybridization.
the results were
0 cells positive
on day 0 and day 3.
counting
we have
as positive.
signal
cells
equivalent
was
yS
color
any
of a
million
result
in sampling
variations
that eliminate
positive
A single positive
cell could be detected
with confidence
sensitivity
of
cell
months,
characterize
low,
possibility
In several
U)
C.)
tion
this
cell
if no
the
many
would
as positive.
negative
over
we
positive
cytoplasm),
is negligible.
viewed
that
levels
sample
3
in the
positive
cells
a cell
background
>
RNA
false
ET AL
cells.
A
correspond
to
value
be
may
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
DETECTION
OF HIV-1-INFECTED
more
compatible
from
Abbott
I cell
erations
per
tion
p24
these
that
cells).
Antigen
situ
by
situ
of lytic
in situ
The
accumulates
Therefore
providing
less
in the
these
it
from
infected
nonisotopic
in situ
distinguishable
Positive
cells
Direct
to
detect
in vitro
as
time
in
p24
as
coculture.
of viral
with
nucleic
and
acids
cells. (This constitutes
individual
after a 7-day
similar
of
a larger
cells
has been
7
cells
days.
this
situ
similar
p24
coculture
cells
with
as positive,
results.
While
with
viral
in
shorter
as well
culture
situ
times
of
as the
of
in situ
improved
x
106)
coculture
of
patient
culture
indicate
more
sensitive
technique
mononuclear
reported.#{176}Furthermore,
produced
both
Dupont
p24 detection
that
time. Preliminary
comparisons
on
that these improvements
increase
samples
scored
as positive.
As described
hybridization
continues
to provide
quanti-
hybridization.
1 6%
an
of
the
(A)
cells
Cells
were
from
positive
a normal
and
lymphocyte
were
clearly
a positive
control
slide used in conjunction
with all our patient
detections.)
coculture
with normal
lymphocytes.
Positive
cells were 0.1 % of total cells.
to (B). Positive
in
in
antigen-capture
hybridization
results.
positive
(10
recently
in situ
point
a 3-week
in
of
the
Results are shown
virus-positive
cells
The
in both
using
have
percent
using
At
the
gave
inoculum
decreased
samples
previously,
in positive
for
hemophiliacs
the
consists
are also expected
to decrease the time needed for
The isolation
of HIV from 98% of HIV seroposi-
with
similar
mononu-
cultured
with
can detect
tive
allows
during
of the
asymptomatic,
24% to 33% of these samples
agreed
and Abbott
in the blood
patient
HIV-1
useful
the ability
detected
samplings
ELISA
but
mothers.
detected
groups
results
HIV-seropositive
one population
T lymphocytes.
culture,’#{176} improvements
virus-positive
HIV
hybridized
cells from patient
cells.)
rare
test
of which
be seen
is to become
the
ELISA
hybridization
SAMPLES
To determine
we
of detection
of positive
of negative
ELISA
should
to detect
from the uninfected
from a seropositive
detection
the contrast
able
individuals.9
infected
the
cells
of both
stimulated
Repeated
increasing
approach
hybridization
Examples
were
with
TO CLINICAL
patients,
WV-infected
Fig 4.
be
with
to 54%
normal,
by
antigen
studied:
T
of
As in the previwith
different
seropositive
.
all
virus-positive
approaches
hybridization
must
of
cells
were
are
coculture.
Two
human
hybridization
all samples
assay.
who
stimulated,
direct
without
of infants of seropositive
1 In situ hybridization
be detected
information.
APPLICATION
clinically,
necessarily
media
two
would
cells
populations
53%
would
normal,
performed
we compared
hemophiliacs
other
Table
with
also
antigen-capture
patient
best
Conversely,
comparable
complementary
If the in situ
not
cells
coculture
and
mononuclear
p24
be emphasized
These
or expression
measurement
becomes
antigen
culture
patient
in
the
after
ous experiment,
replica-
to
It should
infection.
but
direct
culture.
of
viral
hybridization.
hybridization
therefore
cells
(less
improvement
relative
cells
lymphocytes
these consid-
undergoing
tests.
clear
for in situ
detectable
From
magnitude
actively
replication
viral
capture.
not
hybridization
antigen-capture
be detected
in
of
cells
in
as a result
undergoing
by
order
an
now available
level
tests measure
different
parameters
of infection.
is detected
in the supernatant
of the cell cultures,
presumably
not
was
uninfected
is
tests
background
experiment
using
available
sensitive
The
100,000
in detecting
when
2299
in this
there
sensitivity
the more
with
or DuPont).
hybridization
than
CELLS
cells were 0.01 % of total cells. (Note that phase microscopy
(B)
(C)
increases
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
2300
SINGER
tative
cellular
information
complementary
to these
other
techniques.
The
of in situ
hybridization
it immediately
makes
blood
lymphocytes.
lation
investigated
to detect
applicable
Cells
from
rare,
to patient
the same
adult
above
were
positive
tion
peripheralpatient
popu-
using
copy
of a single
HIV
other
of examination
tested
directly
blot,’
number
positive
(PCR),2325
there
approach
described
without
cells
use ofcoculture
detected
cells
per
capture
The
(34%).
in these
well
assay
varied
50,000
on
positive
cells
no obvious
The number
patients
(average
was
CD4-positive
but
coculture
patients
was found
cytes
cells.
and number
of viral-infected
Figure 4 shows representative,
HIV
nucleic
acids
in patient
marrow,
bone
and
fluid
evaluation
of direct
tive patient
preparation
samples
CSF
marrow
of other
samples
cultured
cell
to have a single
cells. Coculture
and
cells.
cells,
by a sample
of
types.
One
a HIV-seropositive
were
were found
cells found
from
cells
also
after
investigated
seroposi-
cell in a direct
patient’s
blood
donors.
their
this
be
used,
reverse
transcriptase
to complete
the assay
gave
large
nor does it require
Several
bone
that
for evaluation
within
tissue
While
the work
just described
is currently
anecdotal,
it serves
to illustrate
further
applications
for this methodology.
Further
increases
in sensitivity
and
convenience
will
of the
of
This
absolute
allows
a
and can be used
If some
patients
could
above,
are
infected
be monitored
vivo).
in
as shown
directly
Second,
the
to detect
infection.26
convenient
virus-
Third,
it procomplement
to
in conjunction
with
to culture
cells. The
in individuals
time required
on patient
be decreased
equipment
specialized
and
It is presumed
the in situ approach
will also prove important
of cytologic
aspects
of the viral
infection
sections
obtained
by biopsy
or autopsy.8’9’2’
or
or p24 assays
7 days.
as well, as
isolated
of the
contribu-
of circulating
culture
or serves as an alternative
with detectable,
circulating
infected
individual
(isotopically);
vitro
to confirm
suspected
rapid,
quantitative,
and
a
reaction
nature
patient
to therapy
(in
to
as dot
in infants
ofseropositive
mothers,
where matermay confuse
the diagnosis;
or in seronegative
positive
cells
nal antibodies
individuals
chain
drugs.
numbers
copy
such
assessment
nucleic
acid.
in the
response
approach
can
approach
load
significant
inoculated
T cells
polymerase
of therapeutic
than
I day and could
requires
no specialized
in these patient
samples
in monocyte
preparations
seropositive
using
samples
is the single-cell
of viral
to contain
vides
a preliminary
infected
of this
Stimulated
of virus-infected
positive
cells
were positive
and
testing
considerably.
from
numbers
mononuclear
mononuclear
in
found
of
or
a single
or complement
of patient
hybridization
evaluation
lymphocytes,
mononuclear
detection
coculture
of in situ
can detect
detec-
are some immediate
applications
in this work. One of the major
to test the effects
lympho-
DNA22
capture,’8
work
for fluorescent
As an alternative
data,
which
allows
a quantitative
numbers
of cells containing
viral
direct
serum.
assessed,
CD4
following
isolated
splenic
10
antigen-
also
between
tions
to
cells produced
a culture
with 100 infected
cells
by day 14 of culture,
increasing
the confidence
of the detection
with
were
cells in culture
infected
(CSF)
were
screened
was found
of spleen
mononuclear
per iO cells
The
patients’
circulating
nonisotopic
by direct detection
using freshly
Peripheral
blood
monocytes,
cerebrospinal
I cell
cells/well).
1 1% of these
of these
correlation
of virus-positive
between
antigen
DNA.
for instance,
developed
of viral
of integrated
means
hybridization;
a protocol
without
coculture.
Approximately
two thirds
of the
of patients
detected
after coculture
were detected
with
in situ
nonisotopic
in progress
ability
cells
improve
ET AL
virus
lymphocytes
considerably.
other than
is less
Finally,
a microscope,
it
training.
ACKNOWLEDGMENT
thank the personnel involved
in handling
Frank Brewster,
Lisa Marselle,
and Maureen
We
ciate
the
use of the
and
Genentech
the
also like to thank
Dupont
their
Tom
Corporation
useful
HIV
help
probe
with
Sharpe,
discussions
blots
Ruth
and
from
by Jim
Barbara
Young,
from
reagents
infectious
Layden.
originally
filter
and Jerry
live
Larry
appre-
Lasky
Monroe.
We
and Carl
Adler
Molecular
for
virus:
We
the
at
would
from
Biosystems
for
probes
and
SNAP
DuPont for help with the p24 assay in our laboratory
(Fig
thank Card Mulder and Harriet
Robinson
for useful advice.
3). We
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1989 74: 2295-2301
Detection of HIV-1-infected cells from patients using nonisotopic in situ
hybridization
RH Singer, KS Byron, JB Lawrence and JL Sullivan
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