Detection of parvovirus B19 DNA in bone marrow cells by chemiluminescence in situ hybridization.
ABSTRACT A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.
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JOURNAL OF CLINICAL MICROBIOLOGY, May 1996, p. 1313–1316
Copyright ? 1996, American Society for Microbiology
Vol. 34, No. 5
Detection of Parvovirus B19 DNA in Bone Marrow Cells by
Chemiluminescence In Situ Hybridization
MONICA MUSIANI,1* ALDO RODA,2MARIALUISA ZERBINI,1GIOVANNA GENTILOMI,1
PATRIZIA PASINI,2GIORGIO GALLINELLA,1AND SIMONA VENTUROLI1
Institute of Microbiology1and Department of Pharmaceutical Sciences,2
University of Bologna, Bologna, Italy
Received 30 October 1995/Returned for modification 16 January 1996/Accepted 15 February 1996
A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in
bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a
highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-
hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical
microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for
image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of
patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with
colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an
objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA
compared with in situ hybridization with colorimetric detection.
Human parvovirus B19 is the etiologic agent of a wide range
of clinical syndromes such as erythema infectiosum, fetal hy-
drops, postinfectious arthropaty, transient aplastic crises in
patients with haemolytic disorders, and chronic bone marrow
failure in immunocompromised patients (1, 4, 26). As parvo-
virus B19 cannot be efficiently grown in established cell cul-
tures, the diagnosis of B19 infection relies mainly on the de-
tection of specific immune response to B19 and/or detection of
B19 genomes by hybridization assays or by PCR (11, 18, 20, 29,
31). In situ hybridization is a successful method for the local-
ization of specific viral nucleic acids inside individual cells, with
the preservation of cellular morphology also permitting retro-
spective studies on archival specimens. In situ hybridization
techniques have been successfully used for the detection of
B19 nucleic acids in bone marrow cells and in fetal tissues
using either isotopic or nonisotopic probes such as biotinylated
or digoxigenin-labeled probes (11, 17, 25, 27, 28). Digoxigenin-
labeled probes have proved very sensitive and specific for the
detection of B19 and other virus genomes in cells or tissue
specimens, giving levels of positive signal similar to those ob-
tained with radioactive probes and proving more sensitive and
specific than biotinylated probes (9, 19, 24).
In in situ hybridization assay, digoxigenin-labeled probes are
immunoenzymatically revealed by using antidigoxigenin Fab
fragments labeled with alkaline phosphatase or with peroxi-
dase followed by colorimetric substrates giving a final colored
product at the site of the enzyme (11). In recent years, chemi-
luminescent substrates have been proposed as a more sensitive
alternative to colorimetric substrates in various analytical tech-
niques in which small amounts of analytes or enzymes have to
be detected (7, 14–16, 22), and they have also been successfully
employed for the search of B19 DNA in dot blot hybridization
assays (13, 23, 30). Moreover, using newly synthesized dioxet-
ane derivates as chemiluminescent substrates for alkaline
phosphatase, we were able to detect as little as 10 fg of B19
DNA in a dot blot hybridization format, with the chemilumi-
nescent substrates proving about 10- to 50-fold more sensitive
than the colorimetric ones (12). Continuing improvements in
chemiluminescent substrates have been matched by new de-
velopments in photon imaging instrumentation such as high-
performance luminographs based on a charge-coupled device
video camera or high-dynamic-range pickup tube (Saticon)
combined with a video amplifier. These instruments not only
allow a quantification of emitted light at the single-photon
level but also permit localization of the chemiluminescent
emission on a target surface. Moreover, by connecting the
luminograph to an optical microscope, it is possible to localize
the light emission inside tissues or cells. All these data
prompted us to explore the use of highly sensitive chemilumi-
nescent substrates and a high-performance luminograph con-
nected to a light microscope for the in situ detection of B19
DNA inside bone marrow cells of patients with aplastic crisis
or hypoplastic anemias using digoxigenin-labeled B19 DNA
probes constructed in our laboratory.
Samples. Bone marrow aspirates from 15 patients with
aplastic crisis or hypoplastic anemias caused by B19, diagnosed
by in situ hybridization with colorimetric detection and dot blot
hybridization or nested PCR in peripheral blood serum sam-
ples were analyzed in the study (2, 11, 20, 21, 31). Dot blot
hybridization was performed with digoxigenin-labeled probes
as previously described (2), and the sensitivity of the assay with
colorimetric detection ranged between 1.5 ? 105and 3 ? 104
genome copies. Nested PCR was performed as previously de-
scribed (20) on peripheral blood samples, treated with a lysis
solution, and the sensitivity of the assay was between 1 and 20
Of the 15 patients, 6 were bone marrow transplant recipi-
ents, 6 were patients with lymphoproliferative disorders under
immunosuppressive therapy, 2 were thalassemic patients, and 1
was an AIDS patient with haemophilia. Moreover, samples
from a bone marrow recipient with hypoplastic anemia who
had proved positive for B19 DNA by in situ hybridization but
negative by dot blot hybridization and nested PCR in periph-
eral blood were also analyzed.
As controls, bone marrow aspirates from 24 patients with
* Corresponding author. Mailing address: Institute of Microbiology,
Via Massarenti 9, 40138 Bologna, Italy. Phone: 39 51 302435. Fax: 39
hematologic disorders who had proved negative for B19 DNA
by in situ hybridization with colorimetric detection, dot blot
hybridization, and nested PCR in peripheral blood were stud-
Bone marrow aspirates from each patient were processed for
chemiluminescent and colorimetric in situ hybridization with
the same probes and in the same run. Samples were analyzed
in duplicate for both chemiluminescence detection and color-
As negative controls cells from the interleukin 3-dependent
leukemic cell line TF-1 were used.
B19 parvovirus DNA probe. The B19 probe was prepared
from the molecular clone of a 5.0-kbp insert (nucleotides 282
to 5310), which represents the complete coding sequence of
B19 DNA, cloned in vector pUC18 (10). Routine methods for
large-scale preparation of plasmids were used. Digoxigenin-
labeling of the probe was performed as previously described
(2) from the excised insert, by the randomly primed DNA
labeling method. The digoxigenin-labeled probe was hybrid-
ized with serially diluted unlabeled parvovirus B19 DNA
bound to nylon membranes, and between 0.5 and 0.1 pg of B19
DNA (corresponding approximately to 1.5 ? 105and 3 ? 104
genome copies) could be visualized by colorimetric detection,
and 10 fg (corresponding approximately to 3 ? 103genome
copies) could be visualized by chemiluminescence detection.
The B19 DNA probe could be stored at ?20?C for at least 1
year with no decrease in its activity.
Bone marrow smear preparation. Bone marrow aspirates
were smeared on slides pretreated as previously described (11)
and were air dried and fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) (0.15 M, pH 7.4) for 10 min.
After fixation, smears were washed three times in PBS for 10
min each and then dehydrated with ethanol washes (30, 60, 80,
95, and 100%) for 5 min each. Smears were then air dried and
stored at 4?C until use.
In situ hybridization reaction. Cell smears were hydrated in
PBS and placed in 0.02 N HCl for 10 min. After three washes
with PBS, cells were treated with 0.01% Triton X-100 in PBS
for 2 min. After three further washes with PBS, cell smears
were treated with pronase (Boehringer, Mannheim, Germany)
(0.5 mg/ml in 0.05 M Tris-HCl [pH 7.6], 5 mM EDTA) for 5
min. Smear preparations were then washed twice with PBS
containing 2 mg of glycine per ml. After these treatments cell
smears were postfixed with 4% paraformaldehyde in PBS and
washed twice with PBS containing 2 mg of glycine per ml.
Smears were then dehydrated by ethanol washes (30, 60, 80, 95,
and 100%). Dehydrated monolayers were overlaid with 10 ?l
of the hybridization mixture. The hybridization mixture con-
sisted of 50% deionized formamide, 10% dextran sulfate, 250
ng of carrier calf thymus DNA per ?l, and 2 ng of digoxigenin-
labeled probe DNA per ?l in 2? SSC buffer (0.3 M NaCl, 0.03
M sodium citrate [pH 7.0]). A clean glass coverslip was then
applied, and the edges were sealed with rubber cement to
prevent loss of the mixture during denaturation and hybridiza-
tion. Cell smears and hybridization mixture were denatured
together by heating in an 85?C water bath for 5 min and were
then incubated at 37?C overnight. After hybridization, the cov-
erslips were carefully removed, and cell smears were washed in
stringent conditions (11).
Chemiluminescence detection. For the detection of hybrid-
ized probes, slides were briefly washed in a 100 mM Tris-HCl
buffer, pH 7.5, containing 150 mM NaCl. Smears were then
incubated for 30 min with sheep polyclonal antidigoxigenin
Fab fragments, conjugated to alkaline phosphatase, and di-
luted 1/500 in blocking reagent (Boehringer). After incubation,
bone marrow smears were washed by two 15-min washes with
Tris-HCl buffer and equilibrated for 2 min with equilibration
buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2[pH
9.5]). The chemiluminescence detection of alkaline phos-
phatase was performed by treating the cells with undiluted
adamantil-1,2-dioxetane phenyl phosphate substrate (CDP-
star) (Tropix, Inc., Bedford, Mass.). After an optimized incu-
bation of 30 min at room temperature, the solution was re-
moved, and the luminescent signal from the hybrid formation
was detected and analyzed with a system which consisted of
luminograph LB-980 (EG&G Berthold, Bad Wilbad, Germa-
ny), which is a high-performance, low-light-level imaging ap-
paratus with a high-dynamic-range pickup tube (Saticon) com-
bined with a video amplifier, connected to a model BH-2 light
microscope (Olympus Optical, Tokyo, Japan) and to a per-
sonal computer with a commercially available program for
image analysis. The microscope was enclosed in a dark box to
prevent contact with the external light. The system operated in
consecutive steps. Firstly, bone marrow cells were recorded in
transmitted light; then, the luminescent signal from the hybrid
formation was measured; and then, after a computer elabora-
tion of the luminescent signal with pseudocolors corresponding
to light intensity, an overlay of the two images on the screen
provided by the transmitted light and by the luminescent signal
allowed the spatial distribution of the target analyte to be
localized and evaluated. Digital images of the light emission
from bone marrow cells were optimized with 2-s integration
intervals for 1 min of total accumulation time. The light emis-
sion from each cell was quantified by defining a fixed area and
summing the total number of photon fluxes per second from
within this area.
As negative controls TF-1 cells were analyzed as described
above, providing threshold background levels. A mean of 50
negative cells were analyzed for each run, and the average
value of photon fluxes per second plus fivefold the standard
deviation was considered the positive threshold for chemilu-
minescence in situ detection of B19 DNA. Corrections for
instrumental background and flat-field variations were auto-
matically performed by the LB-980 apparatus.
To assess the specificity of the chemiluminescence assay, a
series of controls were performed: (i) B19-positive bone mar-
row cells were hybridized with the unlabeled B19 DNA probe;
(ii) B19-positive bone marrow cells were hybridized with the
unlabeled B19 DNA probe; (iii) B19-positive bone marrow
cells were hybridized with the digoxigenin-labeled B19 DNA
probe, but the incubation with anti-digoxigenin antibody was
replaced with an incubation with nonimmune sheep serum;
and (iv) B19-positive bone marrow cells were hybridized with
the digoxigenin-labeled B19 DNA probe, but the incubation
with antidigoxigenin antibody was omitted. The light emission
from control experiments was analyzed and evaluated as de-
Colorimetric detection. Hybridized bone marrow cells were
treated with the colorimetric alkaline phosphatase substrate as
previously described (11), and the development of a dark blue
precipitate at the enzyme site in positive cells was monitored
by microscopic examination.
Of the 15 bone marrow aspirates from patients who had
been proved positive for B19 DNA both by in situ hybridiza-
tion and by dot blot hybridization and/or nested PCR, all 15
samples proved positive by chemiluminescence in situ hybrid-
ization (Fig. 1). When a comparison between the numbers of
cells found positive by chemiluminescence in situ hybridization
and by colorimetric in situ hybridization was made, after the
samples from the same patient had been processed in the same
run and with the same batch of probe, the two methods showed
a high correlation (P ? 0.0005; Spearman rank correlation).
1314NOTESJ. CLIN. MICROBIOL.
With the chemiluminescence method a mean of 19.8 positive
cells per 100 counted cells could be found versus the mean of
4.6 positive cells per 100 counted cells obtained with the col-
orimetric method. This difference was highly significant (P ?
0.0007; Wilcoxon signed test for paired data).
The bone marrow samples from the patient with hypoplastic
anemia who had been proved positive by in situ hybridization
with colorimetric detection but negative by dot blot hybridiza-
tion and nested PCR in peripheral blood was confirmed pos-
itive by chemiluminescence in situ hybridization.
Of the 24 bone marrow aspirates from patients who had
previously proved negative for B19 DNA by in situ hybridiza-
tion with colorimetric detection, by dot blot hybridization, and
by nested PCR in peripheral blood, 23 samples proved nega-
tive by both chemiluminescence in situ hybridization and col-
orimetric in situ hybridization. One sample, however, proved
positive by chemiluminescence in situ hybridization but nega-
tive by colorimetric in situ hybridization. The sample was from
a patient with a chronic anemia after bone marrow transplan-
tation. To establish whether this sample was a false positive, we
tested the sample with another B19 DNA probe of 700 bp that
we had used in previous studies (2, 11, 23, 31), and the positive
result obtained by the chemiluminescence in situ hybridization
and the negative result obtained by the colorimetric reaction
were confirmed. As the peripheral blood from this patient
when retested with nested PCR was confirmed negative for
B19, the nested PCR was performed directly on bone marrow
cells, and a clear positive result was achieved.
A series of control experiments definitely proved that the
chemiluminescence hybridization reaction was detecting par-
vovirus B19 sequences specifically. In fact, no chemilumines-
cent signal was observed when B19-positive bone marrow cells
were hybridized with the plasmid pUC18 control DNA labeled
probe and treated with antidigoxigenin Fab fragment conju-
gated with alkaline phosphatase and with the chemilumines-
cent substrate. Similarly, no luminescent signal was detectable
after hybridization with unlabeled probes followed by the im-
munoenzymatic chemiluminescence treatment. Moreover, B19-
positive bone marrow cells were completely negative after hy-
bridization with the B19 labeled probes when the primary
incubation with antidigoxigenin antibody was either omitted or
replaced with incubation with nonimmune sheep serum.
In recent years there has been a growing interest in the
application of in situ hybridization for the diagnosis of viral
diseases, especially for those viruses, like parvovirus B19, that
cannot be diagnosed by isolation procedures. The possibility of
using a nonradioactive label, such as digoxigenin, which has a
sensitivity similar to that of radioactive labels and a better
resolution, has made this technique more attractive for diag-
nostic laboratories trying to avoid problems related to the short
life of radioactive compounds, disposal, and personnel safety.
In this study we have developed a chemiluminescence in situ
hybridization assay for the search of B19 DNA which could
combine the specificity of digoxigenin-labeled probes, the sen-
sitivity of alkaline phosphatase chemiluminescent substrates,
and the spatial morphological resolution of in situ hybridiza-
tion, using a high-performance, low-light-level imaging lumi-
nograph connected to a light microscope and to a computer for
image analysis. Although the technological equipment em-
ployed in this work is mainly a research tool at this time, in our
study we tried to explore its potentiality for diagnostic pur-
poses. In our assay we used digoxigenin-labeled probes which
were immunoenzymatically revealed by using antidigoxigenin
Fab fragments conjugated with alkaline phosphatase. The use
of Fab fragments in the detection of hybridized probes can be
highly specific without any background due to the presence of
cells expressing Fc receptors, and the use of alkaline phos-
phatase avoids many of the disadvantages of horseradish per-
oxidase, such as interference from endogenous peroxidase ac-
tivity in cells and the need to pretreat cells to inhibit the
enzyme. The chemiluminescence detection of the alkaline
phosphatase-labeled antibody was performed with the 1,2-di-
oxetane substrate CDP-star, which represents one of the most
sensitive detection systems, being able to reveal as few as 1.6
zeptomoles of the enzyme (3); moreover, CDP-star has glow-
ing kinetics with a steady-state emission which permits easier
handling of the specimens, and, since the signal intensity is
proportional to enzyme activity or concentration, it also allows
accurate analysis of the samples. In our chemiluminescence in
situ hybridization assay, signal acquisition was done after the
liquid film of the substrate over the section was removed. This
permitted a sensitive and sharp topographical localization of
the signal, as the mobility of the luminescent product within
the liquid film was avoided and the path and scatter of the
photons were as limited as possible. Moreover, processing the
images by using an image superposition function avoided the
problems which may arise from the sequential analysis of the
FIG. 1. Chemiluminescence in situ hybridization revealing B19 parvovirus in
bone marrow cells. From top to bottom: live image, luminescent signal, and
overlay of the live image and luminescent signal.
VOL. 34, 1996 NOTES 1315
tissue structure and the chemiluminescent signal obtained on
the screen in turn (15).
A sharp topographic distribution of the probe within the cell
was achieved, as the LB-980 instrumentation with the conven-
tional setup has a spatial resolution of 240 ?m, i.e., one pixel
corresponds to this value, but once the instrument is connected
to the microscope and with the use of the 40? and 100?
lenses, pixel size is reduced to 1.1 and 0.41 ?m, respectively.
Our chemiluminescence in situ hybridization to detect B19
DNA in bone marrow cells proved very specific and very sen-
sitive, as all the positive samples from patients with a diag-
nosed B19 infection also proved positive with our assay, with a
larger number of positive cells per sample thus permitting an
easier evaluation of the sample. Moreover, the sample which
had proved positive by in situ hybridization with colorimetric
detection, from a patient who had tested negative for the
presence of B19 DNA by dot blot hybridization and nested
PCR in peripheral blood, was confirmed positive by chemilu-
minescence in situ hybridization.
Moreover, among 24 samples from patients with hematolog-
ical disorders who had proved negative for B19 DNA by in situ
hybridization with colorimetric detection, dot blot hybridiza-
tion, and nested PCR in peripheral blood, chemiluminescence
in situ hybridization confirmed the negative result for 23 sam-
ples and for 1 sample gave a positive result, proving as sensitive
as nested PCR performed on bone marrow cells. Our chemi-
luminescence in situ hybridization thus offers the possibility of
diagnosing B19 infection in a small percentage of the total
number of cells without extraction of viral nucleic acids and
obtaining information on the molecular aspects of B19 infec-
tion, with wide applications for both research and diagnostic
purposes. As the ability of B19 to persist in bone marrow cells
over long periods of time in the absence of detectable viremia
has been demonstrated by PCR directly on bone marrow as-
pirates (4, 6, 8), our chemiluminescence in situ hybridization
could also be useful for monitoring persistent replication of
B19 in bone marrow cells in chronic infections.
The skillful technical help of Marinella Plazzi is gratefully acknowl-
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1316NOTESJ. CLIN. MICROBIOL.