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thesis entitled
RAPID DETECTION OF CYTOMEGALOVIRUS IN HUMAN LUNG AND

BONE MARROW TISSUE SECTIONS BY IN SITU
‘ HYBRIDIZATION TECHNIQUES WITH A
COMMERCIALLY AVAILABLE
DNA PROBE

presented by

Hsiao-Hua Lin

has been accepted towards fulfillment
of the requirements for

Master Clin . Lab . Sci .

 

 

degree in

Major professon

Date December , 1987

 

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RAPID DETECTION OF CYTOMEGALOVIRUS IN HUMAN LUNG AND
BONE MARROW TISSUE SECTIONS BY IN SITU
HYBRIDIZATION TECHNIQUES WITH A
COMMERCIALLY AVAILABLE

DNA PROBE

BY

Hsiao-Hua Lin

A THESIS

Submitted to
Michigan State University
in partial fullfillment of the requirements
for the degree of

MASTER OF SCIENCE

Medical Technology Program

1987

ABSTRACT

RAPID DETECTION OF CYTOMEGALOVIRUS IN HUMAN LUNG AND
BONE MARROW TISSUE SECTIONS BY IN SITU
HYBRIDIZATION TECHNIQUES WITH A
COMMERCIALLY AVAILABLE
DNA PROBE

By
Hsiao-Hua Lin

A c-DNA probe from ENZO BIOCHEM was used to elucidate
Cytomegalovirus infection on formalin-fixed, paraffin-
embedded human lung and bone marrow sectiions by in gigg
hybridization techniques. The kit protocol was modified to
circumvent problems. The specificity of the probe was
evaluated using tissue samples that were determined to be
either positive or negative for CMV based on clinical and
laboratory data. Generally speaking, the probe is fairly
honest in terms of supporting the diagnosis of human
Cytomegalovirus infection in lung tissue. A brick red
inclusion indicates a positive reaction which is easily
differentiated from negative cases. However in bone marrow
samples, it shows nonspecific staining for white blood cells

in relatively fresh-prepared tissue blocks.

This is dedicated to my parent for their love and care.

iii

ACKNOWLEDGMENTS

I would like to thank my major adviser, Dr. Douglas
Estry, for the outstanding job he has done in providing me
with encourgement and advice. I am also indebted to Dr.
Estry and Dr. Joan Mattson for providing laboratory space,
patient specimens and reagents to allow this project to be
completed. I would like to thank my committee, Martha
Thomas, for her assistance in accomplishing the requirements
for my Master's degree.

Special thanks to Dr. Tom Bell at Animal Health
Diagnositic Laboratory at Michigan State University for his

technical assistance.

iv

TABLE OF CONTENTS

Page

LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . 1
History of Cytomegalovirus . . . . . . . . . . . 1
Chracteristics of HCMV . . . . . . . . . . . . . 2
Diagnostic Methods . . . . . . . . . . . . . . . 4
Virus isolation . . . . . . . . . . . . . . . 4
Serological tests . . . . . . . . . . . . . . 5
Microscopic examination . . . . . . . . . . . 7
Nucleic acid Hybridization . . . . . . . . . . . 8
Steps for creating a probe . . . . . . . . . . 8
Basic assay techniques . . . . . . . . . . . . 11
METHOD AND MATERIAL . . . . . . . . . . . . . . . . . . 15
Introduction of the CMV Probe being Evaluated . . 15
Glass Slide Pretreatment . . . . . . . . . . . . 16
Tissue Sectioning . . . . . . . . . . . . . 17
Deparaffinization and Rehydration . . . . . . . . 17
Tissue Section Pretreatment . . . . . . . . . . . 17
Hydrogen peroxide incubation . . . . . . . . . 17
Proteinase K digestion . . . . . . . . . . . . 17
Hybridization Assay . . . . . . . . . . . . . . . 18
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 20
Pretreatment of the sections . . . . . . . . . . 20
Proteinase K digestion study . . . . . . . . . 20
Hydrogen peroxide Treatment . . . . . . . . . 23
Modification of the Kit Protocol . . . . . . . . 25
Heating process . . . . . . . . . . . . . . . 25
Counterstaining . . . . . . . . . . . . . . . 26
Specimen mounting . . . . . . . . . . 26
Reaction Patterns and Specificity . . . . . . . . 26
Staining patterns . . . . . . . . . . . . . . 28
Nonspecific staining . . . . . . . . . . . . . 28

DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O 38
APPEDIX . . . . . . . . . . . . 46
Appendix I: Instruction for Hybridization (CMV) . 46
Appendix II: Instruction for Hybridization (EBV) 47

BIBLI OGMPHY O O O O O O O O O O O O I I O O O O O O O 4 9

LIST OF TABLES

Table Page

I Cloning Protocols Used in Constructing Nucleic 9
Acid Hybridization Probes

II Brief Discription of Patient History 16

III Varying Durations of Time and Concentrations of 23
Proteinase K Treatment

vi

Figure

9-10

11-14

15

16

17

18

19

20

21
22

23

LIST OF FIGURES

Insufficient Proteinase K Digestion of Lung
Adequate Proteinase K and H O Treated Lung
Adequate Proteinase K Digesiign of Negative Lung
Endogenous Peroxidase-like Activity of Bone
Marrow due to Insufficient H O incubation

Bone Marrow Section Incubate: Twice with 3% H 0
Bone Marrow Counterstained with Fast Green 2 2
Human Lung Counterstained with Fast Green

Human Lung without Fast Green Counterstaining
Chracteristic Staining of CMV in Lung

Predominantly Cytoplasmic and Diffuse Staining
in Bone Marrow Specimens

Bone Marrow Tested without Probe

Bone Marrow Tested without Probe and Detection
Complex

Old Infectious Mononucleosis Specimen Tested
with EBV Probe

Old Infectious Mononucleosis Specimen Tested
with CMV Probe

Old Erythroid Hyperplasia Specimen Tested with
CMV Probe

Old Erythroid Hyperplasia Specimen Tested with
EBV Probe

Negative Bone Marrow Tested with EBV Probe
Negative Bone Marrow Tested with CMV Probe

Denaturation Curve of Duplex DNA

vii

Page
21
21
22

22

24
24
27
27
29

30

33

33

34

34

35

35

36
37

4O

LITERATURE REVIEW

History of Cytomegalovirus

Human Cytomegalovirus (HCMV) was first described in
1904 by Ribbert (Ribbert, 1904) as large ”protozoan like"
cells in the kidney of an alleged leutic stillborn. In 1907,
waenstein (waenstein, 1907) described cytoplasmic as well
as intranuclear inclusions. After submitting their findings
to various zoologists for opinions, they concluded that the
inclusions represented protozoa, i.e; coccidia, sporozoa
(gregarines), or amoebas. This idea was refuted by Lange
(Lange, 1922) and Mueller (Mueller, 1922), who reported
seeing cytomegalic inclusions in the kidney cells of
infants. They pointed out that since the inclusions could be
observed in stillbirths, they were probably present before
birth. As protozoa could not pass the placental barrier this
possibility was ruled out. Later investigators continued to
find similar inclusions that were constantly and
characteristically associated with lesions produced by
herpes virus. They referred to this as cytomegalic inclusion
disease (C.I.D.).

Since the cytomegaloviruses are largely species

specific, and human CMV does not grow in experimental
animals, the isolation of CMV was not possible until human
cells could be routinely grown in cultures (Enders et a1,
1949). This work was done by three different groups, Smith
(Smith, 1956), Weller et a1. (Weller, 1957), and Rowe et a1.

(Rowe, 1956).

Characteritics of Human Cytomegalovirus

According mainly to its viral morphology, HCMV
(Betaherpesvirinae) is a member of the Herpesviridae family.
This family includes two other subfamilies representing the
herpes simplex virus (Alphaherpesvirinae) and the
lymphoproliferative virus groups (Gammaherpesvirinae) (Ho,
1982). It replicates in the nucleus of its host cell where
fibers of viral DNA are seen to penetrate into protein
capsids thus forming nucleocapsids (Haguenau, 1975). These
nucleocapsids of icosahedral symmetry 'bud' through the
internal nuclear membrane from which they acquire an
envelope. The virion has a density of 1.20 - 1.21 g/cm3
(Kim, 1976). An amorphous protein material (the tegument) is
found between the envelope and the nucleocapsid. The
inclusion body may have the diameter of almost half of the

nucleus. Ultrastructural studies have shown that the nuclear

inclusions comprise condensed, structurally altered

chromatin, intimately associated with enveloping virions.
Basophilic smaller cytoplasmic inclusions are also present,
but these are of more variable distribution and
conformation. They appear to be associated with assembly of
the complete virion within the cytoplasm so range from
particles that probably represent viral protein coat to
naked nucleoids to complete viral units (Robbins and Cotran,
1974). Like all members of this family, HCMV is unique among
DNA viruses in that its genome contains repeating sequences
(Somogyi, 1986). The entire sequence of HCMV has been cloned
in cosmids (Fleckenstein B, et al, 1982; Demarchi, 1981).

In light of its importance as a pathogen causing a
wide spectrum of clinical disorders, this virus has received
increasing attention in recent years. Infections with HCMV
are the largest single cause of congenital malformation
today. Six to 7 % of all infectious mononucleosis is due to
HCMV (Lamberson, 1985). Infections with this virus may also
constitute a serious problem for immunosuppressed
individuals such as cancer patients, recipients of organ
transplants and acquired immunodeficient patients (H0, 1975-
1977; Lonnqvist, et at, 1984). Primary infection with CMV is
usually not diagnosed because it is asyptomatic or
associated with a mild influenza-like illness in the normal

host. Symptomatic primary CMV may occur in surgical patients

who acquire CMV from blood transfusions and is one of the
causes of the post cardiotomy syndrome after open heart
surgery (Doerr, et a1, 1985). Like other herpesviruses, CMV
causes latent infection. The reactivation of latent CMV in
immunocompromised patients is common but it is often
difficult to be certain that the illness is due to CMV
because of the nonspecific nature of the clinical
manifestation of CMV infection. Under certain laboratory
conditions, HCMV can transform or immortalize embryonic
hamster and human fibroblast cells, a capacity it shares
with two other human herpesviruses, herpes simplex and
Epstein Barr viruses (Thapa, et al, 1985). In addition to
this, HCMV plays an as yet undetermined role in transformed
lymphocytes. Both of these have been implicated to a greater

or lesser degree in human malignancies (Evans, 1976).

Diagnostic Methods

(1) Virus isolation :

The shedding of CMV from the throat, and especially in
the urine, may continue for several weeks and even months
after the acute illness has subsided. In contrast to urine
and throat specimens, CMV can be recovered from blood only
during the acute phase of infection (Colimon et a1, 1984).

After inoculation of the specimen into human diploid cell

cultures, up to four weeks may be required before the
cytopathic effects of CMV are detected and this depends upon
the initial amount of virus in the sample (Panjwani, et a1,

1985).

(2) Serological tests :

Because of the fastidious growth characteristics of
the virus, blood cultures may be negative despite CMV
infection. Therefore, serological studies are recommended in
addition to CMV cultures. A fourfold or greater increase in
antibody level between acute and convalescent phase sera
collected two to three weeks apart is evidence of CMV
infection. Such an increase in antibody titer may occur even
though blood and urine cultures are negative (Smith, 1981).

Differentiation of sera positive and negative for
antibodies to CMV has historically been carried out by the
complement fixation (CF) test. However, the CF test has
several disadvantages: some subclasses of immunoglobulin G
(196) do not fix complement (Haikin, 1980); certain sera
show anticomplementary activity; and the test procedure
itself takes two days. A second test, the indirect
fluorescent antibody (IFA) technique, has been very useful
for the detection of CMV IgG (Chiang, 1970). It is more
sensitive and rapid than the CF test. The indirect

peroxidase antibody (IPA) technique that has been developed

recently (Gerna, 1976) is as rapid and sensitive as IFA and
requires a light microscope instead of a fluorescence
microscope. However, IFA and IPA techniques both have the
problem of nonspecific cytoplasmic staining attributed to
the presence of Fc receptors induced by CMV infection
(Sakuma, 1977). The enzyme-linked immunosorbent assay
(ELISA) is a highly sensitive technique (Engvall, 1971) that
is simple and permits use of stored antigen (Re, et al,
1980). The most sensitive antibody assay technique is
generally acknowledged to be radioimmunoassay (RIA). The
development of RIA for mass screening of HCMV infection
(Kimmel, 1980) has allowed testing of large numbers of
samples with small amounts of serum.

Primary infection may also be diagnosed by
demonstration of CMV-specific IgM in a single serum sample.
The indirect fluorescent method was the first technique used
for detection of CMV IgM antibodies. However, rheumatoid
factors in human sera often give rise to false-positive
results. Elimination of this effect can be accomplished by
separation techniques such as column chromatography and
ultracentrifugation, although the handling of large numbers
of specimens is laborious (Grint, et a1, 1985). With respect
to recurrent CMV infections, it appears that IgM is only

detected in some of these cases and, therefore, its

appearance cannot be relied upon as an indication of
reactivated or recurrent infection (Kangro, 1980). However,
by the use of ELISA, RIA and immunoperoxidase assays for the
detection of CMV-IgA antibodies, diagnosticians were able to
detect recurrent infection in kidney transplant patients, in
many cases, before the CF titer rise was apparent (Sarov,

1980 -1983).

(3) Microscopic methods :

In the past, cells in the urine sediment have been
examined microscopically for CMV inclusions. However,
because of the low sensitivity of this method, it has been
discontinued. Two groups of investigators have recently used
electron microscopy (EM) to rapidly detect the presence of
CMV in infected infants (Lee, 1978; Henry, 1978; Martin, et
al, 1984). Both groups detected virus in the urine, and Lee
et a1, were able to detect CMV particles from oral specimens

as well.

Nucleic acid hybridization analysis --- New techniques

One aspect in clinical microbiology practice has not
significantly changed in the elucidation of specific
etiologic agents since the 18803. Both in the 18803 and
19808, the laboratory required agar-based media to yield the
microorganism before analysis. Nucleic acid hybridization
techniques and monoclonal antibody technology may
potentially eliminate the need for growth of many microbes
and thus reduce the time for identification (Engleberg, et

a1, 1984: Molden, 1986).

Steps for creating a probe

The five essential steps in the production of a
nucleic acid hybridization probe test or monoclonal antibody
analysis are listed in Table I (Edberg, 1986). The discovery
of restriction endonucleases was the critical event that
allowed scientists to fragment nucleic acid for specific
analysis. Large numbers of microbial pathogens are grown and
the nucleic acids are removed from them by the combination
of physical (sonication) and chemical (detergent and alkali)
means. After hydrolysis with restriction endonucleases, the

small pieces of nucleic acid are separated from each other

Table I : Cloning Protocols Used in Constructing
Nucleic Acid Hybridization Probes

 

Cloning Protocol Step

1. Selection of template

2. Insertion of template

3. Seletion of active
template

4. Amplification of
template product

5. Selection of clones

with desired properties

DNA Hybridization

Restriction Endonuclease and
capture of DNA fragments

Ligation to a plasmid-
insertion in recipient
Escherichia coli

Successful insertion shown
by antibiotic sensitivity

Growth of vector in
Escherichia coli

DNA hybidization

 

using electrophoresis on gel and column chromatography.

Based on size distribution and other parameters (Grillner,

et al, 1984), the investigators can choose fragments as

probe to insert into a vector for amplification. The vector

is grown inside a properly selected host cell in large

numbers so that billions of copies of the original

fragment(s) are made. Investigators have to examine each

clone until one is found that has pathogen-specific nucleic

acid processing the right properties for nucleic acid

hybridization analysis. After a clone having the specific

DNA fragment is selected, it must be grown in large

quantities, removed from the host cell and labelled.

10

Traditionally, labelling occurred by a nick translation
process. Single-stranded nicks are introduced into double-
stranded DNA using DNase I. These nicks are subsequently
repaired utilizing a DNA polymerase in the presence of
radioisotope-labelled nucleotides (Mckeating, et al, 1985;
Rigby, 1977). A procedure designed to replace radiolabelled
nucleic acid was first sucessfully developed by Ward and
associates in 1981 (Leary, 1983). They covalently linked
biotin to the pyrimidine ring through an allylamine linker.
It was found that the attachment of biotin to the nucleic
acid did not affect the association properties of the
hybridization probe. The assay itself uses the affinity of
biotin for the egg white protein avidin. This protein has a
dissociation constant for biotin of Kd = 10-15, and four
binding sites for biotin. Avidin can be labelled with an
enzyme or fluorescent probe. When the enzyme-linked avidin
attaches to biotin, a substrate for the enzyme can be added
resulting in the development of color (Bayer, 1980; Langer,
et al, 1981). A second strategy to label nucleic acid
nonradiometrically is the introduction of a hapten directly
onto a pyrimidine ring. Tchen and colleagues (Tchen 1984)
found that guanine residues could be labelled with N-
acetoxyl-N-2-acetylaminofluorene (AAF) and its 7-iodo (AAIF)

derivatives. In practice, the probe consists of nucleic

11

acids to which hapten had been attached. When the probe
interacts with its target, antibody to the hapten can then
be added to the reaction mixture and the end point can be

measured by any secondary antibody technique.

Basic assay techniques

After the nucleic acid has been captured from the
microbial pathogen, inserted into a vector, amplified in a
host, and labelled, the assay method itself is the last step
in the diagnosis of an infectious disease using a nucleic
acid hybridization technique. There are four basic assay
techniques in use. The most extensively studied is the dot,
or dot-blot, hybridization method, which employs a
nitrocellulose filter paper support. Nucleic acid of the
infectious microbe from the patient sample is released and
then bound to a spot on the nitrocellulose filter by a
combination of physical and chemical means (Bittner, et al,
1980). The labelled hybridization probe is added to the spot
under a predetermined set of stringent conditions. If there
is no complementary nucleic acid in the spot, the
hybridization probe will not be bound and will be washed
away during a rinsing step. If there is complementary
nucleic acid from the patient sample, then the nucleic acid

hybridization probe will be bound. A substrate to the label

12

on the nucleic acid hybridization probe is added and the
reaction is determined as positive or negative. The second
major technique is the sandwich hybridization procedure.
This method uses the production of two noncomplementary
nucleic acid sequence reagents. These reagents are produced
from adjacent sites on the gene of the microbial pathogen.
The first nucleic acid sequence is immobilized onto the
solid nitrocellulose support and serves as a target sequence
to interact with nucleic acid sequences from the microbial
pathogen. The second sequence is labelled and serves as the
nucleic acid detector probe. Like dot hybridization, nucleic
acid is first extracted from the clinical sample. The
extracted nucleic acid is mixed with a probe nucleic acid
and hybridized against a target sequence that has been bound
to the nitrocellulose paper. If the microbial pathogen
nucleic acid is complementary, a sandwich will be formed and
the complex will be detected by the addtion of a substrate
to the probe label (Virtnen, et al, 1982). A third method is
the Southern blot technique. This method, described in 1975
(Southern, 1975; 1980), has become the standard by which the
specificity of the nucleic acid sequence is established.
After endonuclease digestion, the mixture is separated using
agarose gel electrophoresis. The DNA fragments are
transferred using either blotting or electroelution to

nitrocellulose paper. The nucleic acid probes are added to

13

the nitrocellulose paper to establish identity. This
technique is primarily utilized to establish identity and is
not amenable to routine clinical testing because of the
large amount of time and reagents required. A fourth
technology, the in gitg hybridization procedure, has the
most applicability to clinical diagnostic pathology
(Marlowe, et al, 1983; Wilkinson, et al, 1986). In this
procedure, human tissue (ie, biopsy, cell culture, etc) is
treated with a combination of diluted detergents, mild
acids, and proteases. This treatment fixes the cells in
their natural configuration while allowing the introduction
of labelled nucleic acid hybridization probes. Analogous to
fluorescent antibody detection of antigens in tissue, 13
situ hybridization allows the direct visualization of
specific DNA sequences as they occur in 3129 (Unger, et al,
1986). This method has proven to be particularly useful for
the detection of viruses from tissue. Myerson and coworkers
(Myerson, 1984) detected cytomegalovirus in open lung biopsy
specimens within 24 hours. In 1982, Chou and Merrigan (Chou
and Merrigan, 1982) reported the detection and quantitation
of cytomegalovirus from human urine specimens utilizing DNA
hygridization with 32P. They were able to detect as few as

10 virus particles per milliliter of urine.

Based on the concepts of DNA hybridization assays,

14

variations can be made by changing the probe synthesis
method, probe length, probe label, assay conditions, sample
pretreatments and methods to immobilize DNA and choose from

them the most suitable assay for each particular use.

METHOD AND MATERIAL

The kit being evaluated was purchased from Enzo
Biochem, Inc., New York, N.Y. and contained a mixture of two
cDNA fragments. The fragments were cloned into the BamHR
site of the vector pBR322. One of them is 13.3 kilobase
pairs, and the other 16.6 kilobase pairs. By nick
translation with the thymidine analog 5-[N-(N-biotinyl-E-
aminocaproyl)-3-aminoally]-deoxyuridine triphosphate the
probe was biotinylated. Strepavidin-biotinylated horseradish
peroxidase was used as the detection complex with
aminoethylcarbazole (AEC) as chromogen. The kit wash buffer
salt consists of 1X PBS, 1% BSA, 0.5m NaCl and SmM EDTA, pH
7.5.

Either cytomegalovirus infected human lung tissue
embedded in paraffin or paraffin embedded CMV infected bone
marrows were used. Five cases of paraffin embedded bone
marrows and one case of lung tissue, negative for CMV based
on clinical criteria were used as negative controls. One
positive lung tissue, two positive bone marrow were used as
positive controls. Tabe II shows a brief description of
patient history, and the patients are assogned a letter for
easy reference and spaces. Some modifications of the kit

protocol have been made according to Forghani's (Forghani,

15

16

Table II: Brief Description of Patient History

 

Patient i

A H85-408
B H87-875
C H87-759
D H87-908
E 887-912
F H86-681
G H68-53

H H68-140

Clinical History

25 year-old white female with a 2 year
history of chronic ulcerative colitis and a
one week history of acute febrile illness;
abnormal lymphocytes were seen on her
admission CBC and the urine CMV titer was
positive (1:128).

65 year-old male with massive
lymphadenopathy, renal shotdown and acute
liver failure. CMV was recovered from the
blood culture.

17 year-old white male with Hodkin's disease,
nodular sclerosis type, diagnosed by excision
of mass on right side of neck.

87 year-old female with multiple myeloma.
white female with mild chronic neutropenia.

81 year-old man, extensive traveler, with
fever of unknown origin. Liver enzymes were
elevated with a non-diagnostic carcinoma in
the past. Serum iron, TIBC and % saturation
were decreased; transferrin was normaal.
Patient died with lymphoma vs virus infection.

Unidentified patient with Erythroid
Hyperplasia.

Unidentified patient with Infectious
Mononucleosis.

 

et al 1985) modification.

To prepare tissue sections for hybridization, glass

slides were first coated with poly-L-lysine to prevent loss

of tissue during the test procedures. Specifically, one

17

milliliter of the poly-L-lysine solution (50ug / ml in
deionized water) was evenly distributed on the surface of
the slide. The slides were allowed to stand for thirty
minutes at room temperature. The excess poly-L-lysine
solution was rinsed off the slides by three successive
washes with deionized water. Following air drying the slides
were ready for use and could be stored at room temperature
for one week. Paraffin embedded tissue was cut using
standard histological techniques and then deparaffinized and
rehydrated by sequential immersion into xylene, absolute
ethanol, 95% ethanol, 80% ethanol and deionized water for
approximately one minute each. The slides were then immersed
into 3% H O in water for five minutes followed by rinsing
in deionized water. This was to remove endogenous peroxidase
activity which might be present in some types of cells. Due
to the large amount of red blood cells (hemoglobin has a
proxidase-like activity), the bone marrow sections were
treated twice with 3% H O . The tissue sections were
digested with 100ug Proéeinase K per ml in PBS by covering
the tissue with solution and incubating for thirty minutes
at 37 C. The reaction was stopped by washing the slide with
5uM EDTA in PBS (wash buffer) for ten to fifteen seconds.
The excess liquid was wiped off the slide and then the

slides were air dried.

18

In gitg hybridization for detection of CMV genomic
material was performed according to the kit protocol, with
only minor modifications. After completing the pretreatment
of the slides, one drop of BIOPROBE (SOLUTION 1,
biotinylated DNA probe) was applied to each specimen and
then covered with a glass coverslip just large enough to
cover the surface of the tissue. Trapping air bubbles under
the coverslip must be avoided. The slides were placed in a
92 C water bath for ten minutes, followed by incubation in a
37 C water bath for thirty minutes. Modification of this
incubation procedure are described in the results. The
coverslip was removed by gently lifting one corner and four
drops of PROBE WASH SOLUTION (SOLUTION 2, 50% deionized
formamide in 0.1x PBS) was applied to each specimen. The
sample was then allowed to stand at room temperature for
seven to ten minutes. The slides were rinsed with WASH
BUFFER using an even stream of solution from a squeeze
bottle for ten to fifteen seconds. The excess moisture was
tapped off the slides and three drops of DETECTION COMPLEX
(SOLUTION 3) were added to each specimen. The slides were
then incubated for ten to fifteen minutes at room
temperature, followed by rinsing with WASH BUFFER for five
to ten seconds. The slides should be left wet while
preparing the solution for the next step. The

chromogen/substrate solution was prepared just before use by

19

removing the nipple from a vial of SOLUTION 4c (100 m mole
acetate buffer, pH7.4) and adding, in the following order,
one drop of SOLUTION 4a (ABC) and mixing gently and then
adding one drop of SOLUTION 4b (H O ) to the vial. This
mixture was mixed well. Four drop: if well mixed
substrate/chromogen were added after the excess moisture was
tapped off the slide and followed by incubation for ten to
fifteen minutes at room temperature. Finally, the slides
were rinsed with a soft stream of WASH BUFFER for five to
ten seconds. Polyvinyl alcohol (Elvanol; Du Pont Co:
Wilmington, Del.) can be used as the mounting medium for
permanent preservation. The slides were evaluated by light

microscopy and a brick red to brown deposit in the cells

indicates a positive reaction.

RESULTS

Pretreatment of the Sections

(1) Proteinase K digestion study :

A series of sections were digested for varying
incubation times and with varying Proteinase K
concentrations to determine the optimal treatment for human
lung tissue. According to guidelines provided by the kit, we
choose 0.01, 0.1, and 1mg/ml as test concentrations and 10,
20 and 30 minutes as incubation periods. Results, shown in
Table III, suggested that incubation of the sections in 0.1
mg/ml proteinase K solution for at least 30 minutes was the
minimum. If proteinase K digestion was not sufficient (Fig.
1), we could not find brick red inclusions as we did with
the appropriate treatment, 0.1mg/ml Proteinase K for 30
minutes at 37 C (Fig. 2-3).

As for bone marrow sections, 0.01, 0.1 mg/ml and 20,
30 minutes were employed as test concentrations and
incubation periods. A positive bone marrow control showed
negative results when the Proteinase K concentration was
less than 0.1 mg/ml or the incubation time was less than 30
minutes. Result showed the false negative reaction on a

positive case when the Proteinase K treatment was not

20

21

 

Figure 1: Insufficient Proteinase K digestion of lung

 

Figure 2: Adequate Proteinase K and H 0 treated lung
22

22

 

Figure 3: Adequate Proteinase K digestion of negative
human lung

 

Figure 4: Endogenous peroxidase-like activity of bone
narrow due to insufficient H O incubation
22

23

optimun. Therefore, we decided to use 0.1 mg/ml Proteinase for 30

minutes at 37 C as the test condition.

Table III: Varying Durations of time and concentration
of Proteinase K treatment for lung tissue.
+: red inclusions were seen in positive
control (Fig.2): 0: red inclusions could
not be found in positive control (Fig.1).

 

 

 

 

0.01 mg/ml 0.10 mg/ml 1.0 mg/ml
10 min 0 0 +
20 min 0 0 +
30 min 0 + +

 

(2) Hydrogen Peroxide Treatment :

Immersion of the specimens into 3% H O in water for 5
minutes was enough to inhibit the endogenoiszperoxidase
activity in lung tissue (Fig. 2). But for bone marrow
sections the five minute incubation still resulted in a red
color in white as well as red blood cells (Fig. 4). This
indicated nonspecific peroxidase-like acitivity in red
cells. An additional five-minute incubation with 3% H O was
added to determine if this eliminated the endogenous 2 2
peroxidase activity from a negative control. Red cell

peroxidase-like activity was successfully removed (shown in

Fig. 5) by this modification. As for the nonspecific

0.-

,.

04" ‘

,.- '\ .'¢ "
J I “ -—r-
"’11. 1 r. c

,- g u. ’7‘, I k

 

Figure 5: Bone marrow incubated twice with 3% H O

 

Figure 6: Bone marrow counterstained with Fast Green

25

staining seen in the white cells, further experiments were
needed to determine whether it was caused by remaining white
cell peroxidase activity or nonspecific binding of avidin to

white cells.

Modifications of the Kit Protocol

(1) Heating Process :

According to the protocol, after application of the
BIOPROBE, the section is to be covered with a coverslip and
placed on a 92 C heating block for 2 to 3 minutes. The
slides usually dried out during this heating process so the
sections fell off while removing the coverslip. In order to
prevent drying of the slide we reduced the heating time to
less than one minute. The results indicated no red
inclusions in a positive CMV specimen. Two additional
modifications were made to solve this problem. One was to
heat the slide by steaming. In this modification, slides
with probe were placed in a petri dish and put on the top of
a beaker containing boiling water. The other method was to
use a 92 C water bath to reduce the water loss during
heating. Either way gave satifactory results. Incubation
time was also modified and the results showed no difference

between a 2-3 minute incubation at 92 C followed by 10-15

26

minute incubation at RT and a 10 minute incubation at 92 C

followed by 30 minute incubation at 37 C.

(2) Counterstaining :

Fast green, which stains cytoplasm, was used as a
counterstain to highlight the red inclusions in CMV positive
specimens (Fig. 6-7). Specimens without counterstaining (Fig.
8) show even more clearly where the inclusions are. By
comparing these two cases of the same area of lung, we found
that counter staining tends to mask the positive reaction
and specimens without counter staining still showed tissue
details. So we decide to leave out this step for better

results and less time and reagent needed.

(3) Specimen Mounting :

Water is suggested by the kit protocol to yield fine
results. But the slides can not be preserved more than one
week as the slides will dry out. A more permanent mounting
media, polyvinyl alcohol, was used to replace water. Figure
1-22 shows a satisfactory result by using polyvinyl alcohol

and the color was not changed after two weeks.

Reaction Patterns and Specificity

27

 

Figure 7: Human lung counterstained with Fast Green

 

Figure 8: Human lung without Fast Green counterstaining

28

(1) Staining Patterns :

CMV replicates in the nucleus of the host cell and
enters the cytoplasm by budding through the nuclear membrane.
Therefore, as we can see in the routine microscopic
examination, the staining pattern of this virus is expected
to be cytoplasmic as well as intranuclear, punctate rather
than diffuse. This characteristic staining pattern can be
shown (Fig. 9-10) in the lung tissue sections tested. As for
most bone marrow cases processed by this protocol, the
staining pattern is predominently cytosolic and diffuse with
only an occasional nuclear or punctate appearance (Fig. 11-
14). This might due to the nonspecific staining problem we
observed for relatively freshly-prepared bone marrow tissue

blocks.

(2) Nonspecific Staining :

The cytoplasmic and diffuse staining pattern of white
blood cells in bone marrow sections which could be shown
even in a negative control case (Fig. 11) indicated the
occurrence of the false-positive nonspecific staining. Two
experiments were performed to rule out the possiblities of
nonspecific avidin binding and remaining nonspecific
peroxidase activity. At first, we left out the probe from
the procedure. No color development indicated there was no

nonspecific avidin from the detection complex bound to the

29

 

Figure 9-10: Chracteristic Staining of CMV in lung

30

 

Figure 11-12: Predominantly cytoplasmic and diffuse
staining in bone marrow specimens

31

v : . ‘L’ "'|'>V‘ . '.Q

A - -p_- 7.... ‘Mk.-_1-sfi

 

Figure 13- 14: Predominantly cytoplsmic and diffuse
staining in bone marrow specimens

32

sample (Fig.15). Streptavidin from another source was also
tested and gave the same results with the detection complex
provided in the kit. Both the probe and the detection
complex were left out to see whether remaining nonspecific
peroxidase alone could develop a color reaction. Figure 16
shows that there was no brick red color development in the
whilte blood cells thus no endogenous nonspecific peroxidase
activity was left in the tissue.

Four more cases of negative control were also tested
against the CMV probe and a probe for Epstein-Barr virus
(EBV) infections. The EBV probe was used as a control. The
staining procedure was similar to that for CMV and it was
felt that if probe specificity was a problem, the EBV probe
would not give the same nonspecific staining seen with CMV
probe. Alternatively, if the nonspecific reaction was due to
interactions of cellular RNA or DNA with the probe vector
(pBR322 in both cases), then both probes would give the same
nonspecific positive result. For case G and H, which are
fairly old bone marrow tissue blocks, we did not get
nonspecific reaction either with CMV or EBV probe. The case
H, diagnosed as Infectious mononucleosis, stained positively
with the EBV probe (Fig. 17) and negative with the CMV probe
(Fig. 18). While case G, diagnosed as Erythroid hyperplasia,
stained negatively with both CMV and EBV probes (Fig. 19-

20). However, two other relatively freshly-prepared bone

 

Figure 15: Bone Marrow tested without probe

 

Figure 16: Bone marrow tested without either probe or
detection complex

34

 

 

Figure 17: Old Infectious Mononucleosis specimen
tested with EBV probe

 

-- ._~.‘ , . . .‘r 9.. I- ...- - -.—; - .>—.~-

Figure 18: Old Infectious Mononucleosis specimen
tested with CMV probe

35

 

Figure 19: Old Erythroid Hyperplasia specimen tested
with CMV probe

 

 

Figure 20: Old Erythroid Hyperplasia specimen tested
with EBV probe

36

marrow blocks, I and J, all stained positively with both EBV
and CMV rpobes indicating a nonspecific reaction with both

probes (Fig.21-22).

 

Figure 21: Negative bone marrow tested with EBV probe

37

 

Figure 22: Negative bone marrow tested with CMV probe

DISCUSSION

Because of the specificity and potentially high
sensitivity of the in gitg hybridization technology, there
has been increasing attention to the application of this
technique in the detection of viral infection. This
methodology recognizes the specific viral genome in a
clinical specimen. In the above study we evaluated a
commercially available biotinylated cytomegalovirus DNA
probe for detection of CMV genomic material in human lung
and bone marrow tissue sections. There are several things
that need to be discussed in this set of experiments. The
first of these is the Proteinase K digestion study. The
Proteinase K digestion procedure is essential for formalin
fixed tissue sections because it exposes the DNA inside the
cells to make the hybridization possible. The exact
concentration and incubation time depends largely on both
the type of tissue being processed and the fixation
condition of the tissue. A series of digestions were
performed to determine the optimal conditions for both human
lung and bone marrow tissue sections. According to the
positive control reactions, 0.1 mg/ml proteinase K
incubation at 37 C for 30 minutes was chosen for both tissue

types without wasting the reagent or time, both critical

38

39

parameters to be evaluated when applying a test to clinical
diagnostic usage.

The heating process which allows the unwinding of the
biotinylated probe DNA as well as target DNA is a second
important topic to be discussed. The denaturation curve of
double-stranded DNA (Fig. 23) indicates that it well become
single-stranded DNA at about 100 C. Prior to this
temperature the amount of single-stranded DNA decreases
rapidly as the temperature decreases. In order to maximize
the percentage of single-stranded DNA for hybridization,
high temperature is required. Formamide (already in the
probe) is added to this reaction to reduce the Tm (melting
temperature) at which DNA unwinds.It is suggested by the kit
protocol that, in order to reach this requirement, direct
heating on a 92 C heating block for 2-3 minutes is required.
Forghani et al. (Forghani et al. 1985) modified this
procedure and used an 80 C water bath and a 10-minute
incubation period. This modification was used to detect
Herpes Simplex Virus DNA in human brain tissue with a probe
purchased from the ENZO BIOCHEM company. Since direct
heating resulted in the tissue sections drying and falling
off the poly-L-lysine coated slides, a shortened incubation
time (less than one minute) was tried to prevent drying of
the slides. This, however resulted in a false negative

reaction. The protocol was then modified by steaming,

40

incubation in a 92 C water bath for 2-3 minutes or 92 C
water bath for 10 minutes. All three incubation protocols
gave the same satisfactory results. Higher temperatures are
not required since they do not cause a significant increase
in the amount of single-stranded DNA but only result in the.‘

slides drying.

A

amount of

single-

stranded
DNA

 

100 C Temp -->

 

Figure 23 : DNA Denaturation Curve. Tm, melting
point of duplex DNA, related to
formamide concentration, G+C content

of DNA and ionic strength of solvent.

41

As mentioned in the results, specimen counterstaining
and mounting were also modified. Counterstaining with fast
green, provided in the kit masked the brick red inclusions
that could be seen clearly without counterstaining. This
procedure was therefore omitted in our modification. As for
mounting fluid, water is very good except for preservation.
Polyvinyl alcohol gave excellent results and provide a much
more permanent mounting media than water. For clinical use
in laboratories that wish to preserve their specimens for a
longer period of time it is suggested that polyvinyl alcohol
be used. Otherwise, use of water is fine and can save money
for reagents.

This probe is fairly honest in terms of
differentiating positive and negative controls in the
control slides provided in the kit as well as in human lung
tissues. The control slides with infected and uninfected
cells placed in different wells were tested against CMV
infection along with other tissue sections. A positive brick
red staining could always be seen in the positive well and
no color reaction developed in the negative well. Positive
and negative lung tissues based on clinical and laboratory
criteria were also tested. Satisfactory results were
obtained. Neither the control slide nor the human lung
tissue has the problem of nonspecific staining. The positive

staining patterns for both of them were intranuclear as well

42

as cytoplasmic.

On the contrary, relatively fresh-prepared bone marrow
samples were stained positively whether they had been
infected with CMV or not. The staining patterns in these
cases were predominently cytoplasmic and diffuse rather than
intranuclear and punctate. Several possibilities were
suggested as causes for this nonspecific reaction.
Nonspecific avidin binding and remaining nonspecific
peroxidase activity were two of them. They were subsequently
ruled out by performing the experiments without probe or
without both the probe and detection complex. If there were
nonspecific avidin binding from the detection complex to the
tissue, the color reaction should develop even with no probe
added. For a similar reason, if there still was nonspecific
peroxidase activity left in the tissue after 3% hydrogen
peroxide treatment, chromogen/substrate solution should be
sufficient for positive color development. We did not get
positive staining in either case which suggested that these
two possibilities were ruled out.

The literature suggested that CMV infection could be
asymptomatic and hence not diagnosed. We suspected our
negative controls were not truly negative. But after
performing two positive, three negative and one suspicious

cases of the relatively fresh bone marrow blocks, it is

43

unlikely that all of them have been infected with CMV. Two
of these negative bone marrow blocks were also tested with
EBV probe and had the same nonspecific staining problem
although not as strong as in the CMV detection. The staining
pattern in these cases were also predominantly cytosolic and
diffuse.

Two relatively old bone marrow blocks, an Infectious
mononucleosis and an Erythroid hyperplasia case, were used
in the CMV hybridization test as negative controls and they
were stained negatively. The same tissues were tested with
EBV DNA probe for the detection of Epstein-Barr virus
infection. The Infectious mononucleosis case stained
positively and the Erythroid hyperplasia case negatively.
This raises at least three questions. 1) Why is the
nonspecific staining only found in freshly prepared bone
marrow blocks and not in relatively old blocks? 2) Does the
predominantly cytoplasmic staining pattern correlate with
the nonspecific reaction? 3) Dose the viral DNA probe or the
bacteria plasmid vector pBR322 have sequence homology with
either native cellular DNA or cytoplsmic RNA? Further
investigations may include trying to find a relatively old
CMV positive bone marrow block and test it against this CMV
probe from ENZO company to see whether there are some
nonspecific factors present in white blood cells that will

disappear during aging as is indicated by the EBV probe.

44

Perhaps old bone marrow blocks are totally nonreactive to
this CMV probe. By comparing the staining patterns between
lung and bone marrow sections, we find that predominent
cytoplasmic staining is only found in relatively fresh bone
marrow blocks. Cytoplasmic RNA is a possiblility for
nonspecific hybridization with the probe. Because of the
unstability of RNA, it would also explain why we did not get
nonspecific staining in relatively old bone marrow blocks.
Pretreatment of the bone marrow tissue sections with RNAse
to remove RNA before hybridization may provide a way to
eliminate this problem. If RNAse digested bone marrow
sections loose the nonspecific cytoplsmic staining reaction,
it would be concluded that RNA accounts for the nonspecific
reaction. Both the CMV and EBV probe from ENZO are cloned
into a pBR322 vector. Ambinder's group (Ambinder, et al,
1986) has suggested that this vector is the cause of their
vector homology problem in diagnositic nucleic acid
hybridization of various types of clinical specimens. They
also emphasize that it is essential that all hybridization
detection systems use a control probe of the vector alone in
order to demonstrate the absence of material with vector
homology in the specimen tested. In addition to this,
several approaches to determine this problem may include the

use of isolated insert probes, alternative cloning vectors,

45

and competitor pBR322 DNA in prehybridization and

hybridization mixes.

APPENDIX

I. INSTRUCTIONS FOR HYBRIDIZATION/DETECTION 0F CMV

1.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Immerse slides sequentially into xylene, absolute
alcohol, 95% alcohol, 80% alcohol and water.

Incubate in 3% hydrogen peroxide for 5 minutes. (once
for lung: twice for bone marrow)

Stop the incubation with water.

Apply 1ml of 0.1 mg/ml Proteinase K solution.

Incubate at 37 C for 30 minutes.

Stop the reaction with PBS-EDTA (5 umole EDTA in PBS).
Apply one drop of probe solution to slide. (VIAL 1).
Cover with coverslip.

Heat on 92 C water bath for 10 minutes or 2-3 minutes.

Hybridize at 37 C for 30 minutes or RT for 10-15
minutes.

Remove coverslip (Rinse with wash to loosen coverslip).
Tap off excess liquid.

Apply 4 drops of probe wash solution. (VIAL 2)

Let sit at room temperature for 7-10 minutes.

Rinse with wash buffer for 5-10 seconds.

Tap excess moisture off the slide.

Apply 3 drops of detection complex. (VIAL 3)

Let sit at room temperature for 10-15 minutes.

Prepare substrate/chromogen solution.

To one vial of acetate buffer (VIAL 4c) add one drop of
chromogen (VIAL 4a) and one drop of substrate (VIAL 4b)

46

20.

21.

22.

23.

24.

25.

26.

II.

10.

47

Rinse slide with wash buffer for 5-10 seconds.
Tap off excess moisture from the slide.

Apply substrate/chromogen solution to slide.
Incubate at room temperature for 10-15 minutes.
Rinse gently with water.

Mount coverslip with polyvinyl alcohol.

View under microscope.

INSTRUCTIONS FOR HYBRIDIZATION/DETECTION OF EBV

Immerse slides sequentially into xylene, absolute
alcohol, 95% alcohol, 80% alcohol and water.

Incubate slide with 3% hydrogen peroxide for 5 minutes.
(one change for lung: two changes for bone marrow)

Stop the incubation with water.

Apply 1 ml of 0.1 mg/ml Proteinase K solution to the
slide.

Incubate at 37 C for 30 minutes.

Stop the reaction by rinsing the slide with 5 umole
EDTA in PBS.

Mix 4ul of carrier DNA with 20ul probe.

Mix following for the hybridization mixture
50 ul deionized formamide (hybridization solvent)
pH6.8 to 7.2
20 ul dextran sulfate (hybridization reaction
accelerator)
10 ul 20X SSC
24 ul carrier-probe mixture

Apply 20 ul of hybridization mixture to the slide.

Cover with coverslip.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

48

Incubate in 92 C water bath for 2-3 minutes (10 min).
Continue incubation at room temperature for 60 minutes.

Remove the coverslip by immersing the slide into 50%
formamide in 0.1x PBS for 5-10 minutes at room temp.

Rinse in 1x PBS-EDTA at room temperature for 5 minutes.
Apply 3 drops of detection complex.

Let sit at room temperature for 10-15 minutes.
Prepare substrate/chromogen solution as follows :
To one vial of acetate buffer add one drop of
chromogen and one drop of substrate. Mix.

Rinse the slide with wash buffer.

Tap excess moisture off the slide.

Apply substrate/chromogen solution to slide.
Incubate at room temperature for 10-15 minutes.
Rinse gently with water.

Mount the coverslip with polyvinyl alcohol.

View under microscope.

NOTE : The hybridization buffer contains formamide. Each

increase of 1% in the formamide concentration lowers
the Tm of a DNA duplex by 0.7 C.

Tm, melting temperature of duplex DNA, related to
formamide concentration, G+C content of DNA and ionic
strength of solvent.

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