JOURNAL OF PATHOLOGY, VOL.
178 11-20 (1996)
REVIEW ARTICLE
IN SITU PCR: PATHOLOGIST’S DREAM OR
NIGHTMARE?
J. J. O’LEARY, R. CHETTY, A. K. GRAHAM AND J. O’D. MCGEE
Nufield Department of Pathology and Bacteriology, University of Oxford, Oxford, U.K.
INTRODUCTION
The polymerase chain reaction (PCR) represents one
of the most exciting developments in molecular pathol-
ogy, but one important limitation is the inability to
visualize and localize the amplified product within cells
and tissue specimens. Solution-phase polymerase chain
reaction (SPPCR), performed in a single reaction tube, is
an extremely sensitive and specific technique, which has
the ability to amplify single copy genes and rare
sequences in tissues, which are then detectable by
Southern blot analysis, dot blot or gel electrophoresis.
For this approach, nucleic acid extraction is first
required, which necessitates cellular destruction before
amplification; correlation of results with histological
features is rarely possible.
In situ hybridization (ISH) permits the localization of
specific nucleic acid sequences at the cellular level with
high specificity, but this is sometimes overshadowed
by a relatively low detection sensitivity. In in situ detec-
tion systems, the threshold level for messenger RNA
(mRNA) is approximately 20 copies per cell and
most conventional non-isotopic ISH protocols, except
for those incorporating elaborate sandwich detection
techniques,’ do not detect single copy genes.
A number of studies have recently been described
which have combined the use of PCR with IS€%?-’These
techniques will soon lend themselves to diagnostic appli-
cations in the investigation of infections and tumours.
Many centres, however, have encountered problems
with the technique, with reaction failure commonly
reported. This review examines the technique, evaluates
currently available protocols, and suggests possible ways
of circumventing problems that may be encountered.
TERMINOLOGY
In situ PCR
This refers to PCR amplification of cellular sequences
(DNA and mRNA) in tissue specimens, using either a
labelled probe or a labelled oligonucleotide, such as
dUTP, within the PCR reaction mix. In sifu reverse
transcriptase (RT) PCR for the amplification of mRNA
Addressee for correspondence: Dr J. J. OLeary, University of
Oxford, Nuffield Department of Pathology and Bacteriology, Level 4,
John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.
CCC 0022-3417/96/010011-10
01996 by John Wiley & Sons, Ltd.
sequences in cells requires copy DNA template (cDNA)
to be first created using reverse transcriptase, and then
amplified as for in situ PCR. The labelled product is then
detected using standard techniques, as for conventional
ISH or immunocytochemistry.
PCR in situ hybridization
This refers to PCR amplification of cellular sequences
(DNA or mRNA) in tissue specimens, followed by in
situ hybridization detection of the amplified product
using a labelled internal or genomic probe. The labels
used can either by radioactive (32P, 35S) or non-
radioactive (biotin, digoxigenin, fluorescein); most
studies to date have utilized non-radioactive labels.
PRINCIPLES OF ZN SZTU PCR TECHNIQUES
In situ PCR and PCR in situ hybridization represent
the marriage of PCR and ISH techniques, allowing the
amplification of specific nucleic acid sequences inside
cells and increasing the copy number of the target
sequence to levels which are easily detectable by ISH or
immunohistochemistry. The principles of in situ PCR
and PCR ISH are outlined in Fig. 1.
Before amplification is undertaken, cells or tissues are
fixed with a suitable fixative (usually formaldehyde-
based) and permeabilized using proteolytic enzymes
in order to maintain cellular morphology and permit
access of PCR reagents into the intracellular compart-
ment. Amplification then occurs at the defined target
intracellular sequence.
PCR amplification of the desired target sequence can
be performed either in intact cells in Eppendorf tubes
(as in SPPCR), or directly in cytocentrifuge preparations
or tissue sections on glass microscope slides, by over-
laying the sections/cell suspensions with PCR mix and
covering with a coverslip. In theory, fixed cells act as
‘semi-permeable amplifying bags’ which permit entry of
primers, oligonucleotides, and Taq polymerase into the
cell cytoplasm and nucleoplasm, yet regard the outward
diffusion of PCR product, allowing visualization by ISH
of the amplified product within the cell. The equipment
used for these techniques has ranged from standard
heating blocks to thermal cycling ovens; specifically
designed in situ PCR thermocyclers have also been
Received 31 May 1995
Accepted 14 July 1995
12 J. J. O'LEARY E T AL.

Pipette PCR reaction mix

onto tissues and cover with
a coverslip.

Following PCR, add biotin or

digoxigenin labelled probe

-----

. I

\
\
\
\

Pipette PCR reaction

mix containing labelled
nucleotide or primer
onto tissues and cover
I
' Develop a s for
conventional in-
situ hybridisation

with a coverslip.

Tissue attached to microscope slides

.
Develop as for
conventional in-situ
hybridisation

Fig. I-Schmatic representation of PCR in situ hybridization (A) and in situ PCR (B). (A) The PCR reaction mix is placed onto
the tissue section and covered with a coverslip. Following amplification, a labelled probe is added and the product is developed
as for conventional in situ hybridization. (B) The PCR reaction mix contains labelled nucleotide or primer, which IS placed onto
the tissue section and covered with a coverslip. Once again, following amplification, the products are developed using standard
immunocytochemical techniques
 IN SITU PCR 13

Fig. 2 4 A ) NISH (non-isotopic in situ hybridization) of fixed SiHa cells showing 1-2 copies of HPV 16 per cell nucleus. These signals
were achieved using a complex five-step immunocytochemical detection procedure as described by Herrington et al.' (B) PCR ISH of
fixed SiHa cells showing amplification at two loci within the nuclei after 25 cycles. Note that product diffusion occurs at one of the loci
in the cell on the right. The arrows indicate the sites of amplification. Note that discrete signals are obtained. (C) PCR ISH of fixed SiHa
cells showing a diffuse product following 40 cycles of amplification. Note the greater accumulation of PCR product following the greater
number of cycles used [cf. (B)]

described, with in-built slide calibration temperature
curves which optimize heat transfer kinetics.
SPECIFIC APPLICATIONS TO DATE
Several groups have used in situ PCR techniques to
identify single copy sequences in single cells and low
copy number DNA sequences in tissue sections. The
majority of studies to date have focused on the detection
of viral DNA sequences in defined systems [human
immunodeficiency virus (HIV), human papilloma virus
(HPV), mouse mammary tumour virus (MMTV), pro-
virus, cytomegalovirus (CMV), hepatitis B virus
(HBV)]2,7-13(Figs 2-5). Clinical aspects of viral disease
have been examined in a few studies, particularly with
HIV, where it has been discovered that a larger number
of cells and cell types are latent1 infected with the virus
than was originally assumed?,' These findings, which
are of particular interest to molecular biologists,
pathologists, and virologists alike, could not have been
obtained by any other existing molecular biological
technique. Some groups have examined endogenous
human DNA sequences, including single copy human
genes, chromosomal rearrangements, and transloca-
t i o n ~ . ' ~ " In
~ , situ
' ~ RT PCR has been applied success-
fully to detect amplified immunoglobulin mRNA
sequences in single cells. l4
The ability to amplify tumour-specific nucleic acid
sequences as possible 'clonal markers' of malignancy has
enormous potential for the future. These include T-cell
receptor gene rearrangements, translocations, and point
mutations. Applications such as these will be invaluable
in diagnostic tumour pathology and may, with refine-
ments of the technique, allow quantitative assessments
of gene rearrangements and other specific features
before and after treatment (see Table I).
PROTOCOLS AND PROCEDURES: A CRITICAL
EVALUATION
Many protocols have been suggested by different
groups working in this area of research; several of these
share key steps (Table 11), although significant method-
ological differences exist. Details of the technique, in
many cases unique to the particular application, appear
to be extremely important determinants of eventual
success.
Starting material and equipment
Set up procedures for in situ PCR and PCR ISH differ
greatly between groups. In situ PCR of fixed, single cells
14 J. J. O'LEARY ET AL.

Fig. 3-(A) In situ PCR amplification of globin gene in fixed SiHa cells showing diffuse amplification in most cells. p globin gene is used
as a positive reference control gene for the reaction to check whether amplification takes place. (B) In situ PCR amplification of /3 globin gene
in fixed SiHa cells, omitting Taq DNA polymerase: no signal visible. This is used to verify that the signal obtained in (A) was due to the action
of Taq DNA polymerase, and not to spurious binding of detection reagents

Fig. &-(A) NISH of HPV 16 in fixed CaSki cells (200-300 copies of HPV 16 per cell nucleus). Note the dot positivity signal within the
nuclei of the cells. (B) PCR ISH of HPV 16 in fixed CaSki cells showing diffusion of product from the site of manufacture. In comparison
with (A), there is increased signal which obscures cellular morphology and detail

suspended in PCR reaction buffer was first described by
Haase et al. in 1990.2They placed suspended cells in an
Eppendorf tube and proceeded as for conventional
SPPCR using a standard thermocycler. Following
amplification, cells were cytocentrifuged onto glass
slides and the amplified product was subsequently
detected using ISH. Another early approach used pieces
of glass slides with adherent cells from cytocentrifuge
preparations in standard Eppendorf tubes. These were
incubated directly in PCR reaction buffer using standard
SPPCR.I3It seems clear from the data reported by these
groups that protocols using intact cells favour successful
amplification and achieve optimal thermal kinetics
within the reaction mix.
More recently, techniques using tissues and cells
attached to microscope slides have been described.P"'
PCR amplification was carried out either on a heating
block or in a cycling oven, neither of which was specifi-
cally designed to perform these techniques. Most inves-
tigators covered a standard multi-well PCR block with
an aluminium foil boat into which the slide containing
the tissue section was placed and overlaid with PCR
reaction mix and mineral oil.
Optimization of thermal conduction must be achieved
if successful amplification is to occur. One of the most
critical parameters in PCR is the attainment of correct
annealing and denaturation temperatures at the level of
the tissue section, where the amplification process
occurs. 'Thermal lag' occurs, with differences in tem-
perature between the block face, the glass slide, and the
PCR reaction mix at each temperature step of the
reaction cycle; this has not been adequately addressed by
many authors. Our own group has examined this phe-
nomenon and found a significant temperature differen-
tial between the sample and the block during the
denaturation, primer annealing, and extension phases of
the reaction. In some cases, this is of the order of 34"C,
which in most cases contributes to total reaction failure,
largely because initial denaturation temperatures are
never really achieved.6
Thermoconduction in most protocols has been maxi-
mized by filling unused air spaces with water or mineral
 IN SITU PCR
Fig. 5-(A) NISH of HPV 6 in benign metaplastic squamous epithelium in an endometrial adenoacanthoma. Note the positive signal in
occasional cell nuclei. This tissue is from formalin-fixed, paraffin wax-embedded material. (B) HPV 6 PCR ISH of benign metaplastic
squamous epithelium in a parallel section from the case described in (A). Many more nuclei are now showing HPV 6 positivity. However,
preservation of cellular morphology once again is poor. This case illustrates that PCR ISH works on formalin-fixed, paraffin-embedded
tissue with demonstration of greater target DNA than NISH
oil, or by placing the glass slide in an aluminium foil
b ~ a t .Alternatively,
~.~ the heating block and glass slides
can be covered with a plastic lid to optimize heat
trapping and humidity. Ideally, a thermocouple should
be used to assess the temperature of the slide, block, and
sample, where possible, during each of the steps of the
amplification process. Recently, machines have become
available which offer in-built slide temperature calibra-
tion curves, which correct for thermal lag phenomena.
Perkin Elmer have introduced their new Gene Amp
in-situ PCR machine 1000@, which corrects for this
problem and which has a specifically designed thermal
block that optimizes thermal conduction from the block
face to the glass slide and thence to the tissue section.
Tissue reparation
For in situ PCR techniques to work, the cytoskeleton
of the cell must firstly be made ‘rigid’ to preserve cellular
morphology and create a micro-environment within the
cell, akin to that of the Eppendorf tube, as for SPPCR.
Table I-Some uses of in situ PCR and PCR in situ
hybridization
Detection of infectious agents
Human papilloma virus (HPV)
Human immunodeficiency virus (HIV)
Cytomegalovirus (CMV)
Hepatitis B virus (HBV)
Identification of chromosomal rearrangements and
translocations
Immunoglobulin heavy and light variable chain genes
B and T clonality studies
t14118 (Bcl-2)
t l 1:22 Ewing’dperipheral neuroectodermal tumour (PNET)
Studies of cellular gene expression
Epidermal growth factor (EGF) mRNA expression
15
It has been demonstrated that not all fixatives facilitate
in situ PCR techniques. In general, successful DNA
amplification has been achieved with tissues fixed in 1 4
per cent paraformaldehyde, neutral buffered formalde-
hyde, and 10 per cent formalin for 12-24 h.12,16
Occasionally, alcohol and acetic acid-fixed tissues may
be amplified,16 but this is not universally accepted.
Our experience, and that of others, is that amplifica-
tion in alcohol and acetic acid-fixed tissues attached to
glass slides appears to occur preferentially in solution
phase (i.e. in the PCR mix overlying the cells/tissue
specimen). Fixation of cells with formaldehyde fixatives
has a number of drawbacks familiar to histopath-
ologists, but not always appreciated by most in situ
PCR investigators. Formaldehyde is not easily removed
from tissues, even after extensive washing. Formalde-
hyde groups react with nucleic acid template to form
DNA-DNA and DNA-histone protein cross-links. l 7
Cross-linked histone proteins are a major obstacle to
progression of Taq DNA polymerase along target DNA
template, particularly when amplifying products of
size 50-200 bp, considering that the average inter-
nucleosome distance is of similar size. This explains
some of the difficulties encountered by those performing
SPPCR, where fixation is a major determining factor
in successful amplification. A final problem with
formaldehyde-fixed tissue is that nicks occur in the DNA
template. Some of these are non-blunt ended and can
subsequently act as potential priming sites for extension
by Taq DNA polymerase even at room temperature,
leading to spurious results.
Once cells are fixed, firm attachment to glass slides
must be achieved; repeated cycles of heating and cooling
during amplification tend to result in detachment. Slides
pretreated with coating agents ensure maximal section
adhesion. The most commonly used coating agents are
aminopropyltriethoxysilane (APES), Denhardt’s solu-
tion, and Elmer’s glue. Cell permeabilization is then
carried out in order to facilitate entry of reagents into
the nucleoplasm of the cell. This is achieved by several
Table 11-Examples of published techniques for in situ PCR and PCR in situ hybridization

Investigator
Nuovo3 Bagasra et a1.' Komminoth et al." Spann et nI.l3 O'Leary et a1.'2 Haase et aL2 Chiu et aL9 Embleton et aI.l4

Cell adhesion Cytospin and Air-dried Cytospin Adhesion slide APES Elmer's glue APES Cell suspension
tissues APES

Cell fixation Buffered 105°C for 2% paraformaldehyde, 2% paraformaldehyde Buffered Buffered Ethanol, 10% formal
formaldehyde 15 s 95% ethanol, formaldehyde, formaldehyde, acetic acid, saline,
10 min 1248h para formaldehyde, 48 h lh
10 min-72 h
- L
Permeabilization Trypsinogen, Proteinase K, HCI 0.2 N, HCI 0.02 N, Proteinase K NaOH 0.5 M, NP-40 0.5%,
NaCl 1.5 M, Ih L
pepsinogen, 5572, 2 h 20 min 10 min, * SDS,
15 rnin 0.1% Triton-X, 15 rnin 20 min
90 s;
proteinase K,
15 rnin 3b
Denaturation P
During cycling During cycling 95% formamide, During cycling During cycling During cycling During cycling During cycling
0.1% ssc,
70°C, 15 rnin
PCR machine Perkin Elmer Gen Res ProOven Perkin Elmer Perkin Elmer BioOven Perkin Elmer Techne
Perkin Elmer BioOven

Product size 115 bp 115 bp 196-640 bp 140-230 bp 120 bp > 1 kb > l kb

[Primer], ,UM 1 1.3 2 2.5 I 1 0.5 0.5

TaqllOO pl 10 6.6 5 1 10 15 10 5

[MgC1,1, mM 4.5 2.5 1.5 1.5 4.5 1.5 2.5 1.5

 IN SITU PCR
methods, including mild protease treatment or mild acid
hydrolysis (0.01-0.1 N HCl) (see Table 11). NUOVO, for
instance, used trypsinogen or pepsinogen with HCl,
which has the potential advantage that the reaction
can easil be terminated by changing the pH of the
reaction.’ In SPPCR, extensive proteolytic digestion,
occasionally for up to 48 h, is employed to overcome the
problem of DNA-DNA and DNA-histone protein
cross-linking. This cannot be performed, however, with
in situ PCR techniques, as extensive proteolysis destroys
cellular morphology. Therefore, incomplete dissociation
of histone protein-DNA cross-links occurs in in situ
PCR techniques. Acid hydrolysis probably acts by
driving such cross-links to complete dissociation.
Problems do arise, however, with proteolytic digestion;
Komminoth et ul. have reported increased diffusion of
PCR products after proteolytic digestion. l o
AmpliJication: reagents and conditions
Setting u p s e t t i n g up PCR involves the careful
choice of target sequence (taking into account its Tm),
the choice of primers (taking into account I”, the ability
to form primer-dimers, and uniqueness), and the careful
optimization of the PCR cycle (annealing temperature,
denaturation temperature, and cycle number). Initial
denaturation of DNA can be achieved either at the
beginning of thermal cycling, or separately during per-
meabilization or following the fixation process. How-
ever, it is advisable to perform the denaturation step
during the cycling procedure. This can be achieved using
heat, heat/formamide, or alkaline d e n a t ~ r a t i o n . ~ - ~ . ’ ~the tissue. Coating slides with 0.1-1 per cent bovine
Standard cycling protocols can be used, but most inves-
tigators advocate the use of ‘hot-start’ PCR to reduce
mispriming and primer oligomerization. Its role, how-
ever, in preventing false positives is uncertain. Nuovo
has also suggested the use of single strand binding
protein (SSB) derived from Escherichiu coli, which is
involved in DNA replication and repair.3 The precise
mode of action is unknown, but it may act by relaxing
regions of double strandedness and making the precise
target sequences amenable to priming. In the hope of
increasing amplification efficiency, most groups have
used PCR mixtures with higher concentrations of
primers, magnesium, and/or Taq DNA polymerase than
for conventional SPPCR.
Volume of reagents-The volume of PCR reagents
varies, depending on the size of the cell preparation/
tissue specimen that one wishes to amplify. In general,
between 15 and 75pl of PCR reagents is used for
most ‘in situ PCR’ techniques that use glass-mounted
material. Small variations in the volume of these
reagents over the slide can give rise to local PCR failure
and contribute to the ‘patchy’ result obtained.
Number of cycles-The optimal number of cycles
depends on the particular assay. The lower the number
of cycles the better, as product diffusion problems are
less likely to be encountered and cellular morphology is
maintained. Most protocols use between 25 and 30
cycles of amplification, exceptionally 50 cycles. Some
17
groups have used two rounds of 25-30 cycles with the
addition of new PCR mix and Taq DNA polymerase, or
nested PCR with two rounds of 30-cycle amplifica-
tion.2.5.7,9.14,18 The level of amplification achieved in any
particular PCR is difficult to assess. Widely contradic-
tory figures are given, with Nuovo3 reporting 200- to
300-fold amplification, while Embretson et aL7 estimate
amplification of the order of 10- to 30-fold, depending
on the number of cycles; our own observations would
tend to support the latter. This represents a poor rate of
return when one considers the increased concentrations
of enzyme and reagents used at the outset. In terms of
amplification per cycle, 10- to 30-fold levels represent an
amplification factor of 1.1-1.2; in real terms, almost
linear amplification.
Sequestration of reagents-For successful amplifica-
tion to occur with formaldehyde-fixed tissues attached
to glass slides, increased concentrations (2-5 times) of
PCR reagents are essential. However, this is not always
required with in situ PCR techniques carried out on
suspended cells in Eppendorf tubes. This is an interest-
ing, yet costly requirement if one considers that the level
of starting target nucleic acid is often less than that used
in SPPCR. The most logical explanation for using such
high concentrations is that some or all of the reagents
are sequestered during the reaction and are not sub-
sequently available for the process. Sequestration of
reagents can occur because of reagents adhering to the
glass slides, the coating reagents on the slides, or direct
intercalation by the residual fixative residues present in
serum albumin (BSA) allows decreased concentrations
of reagents to be used; BSA may act by blocking
sequestration of reagents. l 9
Primer selection-Primer selection has evolved
around two basic strategies: single primer pair^^.'^'^.'^
or multiple primer pairs, with or without complemen-
tary tails.53,9,14 The multiple primer pair approach
was designed to generate longer and/or overlapping
product, with less potential to diffuse from the site of
manufacture.
Evaporation-PCR mix evaporation can be
adequately prevented by performing amplification in
welled slides, covering the PCR reactants with a
coverslip and then sealing the reaction ‘chamber’ with
nail varnish. Overlaying with mineral oil also helps to
minimize evaporation of reactants.
Diflusion-Diffusion of product from the site of
manufacture is also encountered (see Fig. 4B). As dis-
cussed above, the lower the cycle number used, the less
likely it is that diffusion will occur, depending on the
amount of starting material in the cell. Post-fixation
with paraformaldehyde and alcohol will sometimes help
to maintain localization of the product. Other strategies
have also been designed to minimize product diffusion
following amplification. These include overlaying the
tissue section with a thin layer of agarose, limiting
the cycle number, or directly incorporating biotin-
18 J. J. O’LEARY ET AL.
substituted nucleotides (as in in situ PCR), which renders
the product more bulky and less likely to diffuse.
Patchy amplijication-Patchy amplification is always
encountered, with on average 30-50 per cent of cells
showing amplification signals for the desired target
sequence.12 There are many reasons for this, including
patchy digestion during cell permeabilization steps. In
addition, lingering fixative-DNA and DNA-histone
protein interactions can also contribute to the patchiness
of the signal obtained in any particular tissue specimen.
Finally, because processed tissues are cut by micro-
tome blades during routine histological preparation, the
tops of many cells are ‘truncated’ with two conse-
quences. Firstly, target sequence may be lost from an
individual cell during cutting and, secondly, ‘truncated’
cells by their nature may not contain the amplified
product and the cell yields a negative result.
Post-PCR treatment
Most in situ PCR protocols include a post-
amplification washing step, mainly in order to remove
diffused extracellular product. The inclusion of such a
step is advisable, as this reduces the chances of generat-
ing a false-positive result. In some studies, post-fixation
with 4 per cent paraformaldehyde and/or alcohol
has been used, but this is one area which still needs
considerable work.
Visualization of PCU product
In general, non-radioactive labels provide degrees of
detection sensitivity similar to radioactive labels. On a
theoretical basis, maximum specificity in the identifica-
tion of PCR product using PCR ISH should only be
achieved by using probes which recognize sequences
internal to the amplified product, a strategy which is
essentially similar to Southern blot/dot blot confor-
mational analysis of product obtained from SPPCR
techniques. However, genomic probes can also be used
which are not restricted to the primer free se-
quences.2.5,7.12,18In situ PCR has also been performed
with radioactive and non-radioactive labels. One advan-
tage of this technique is that post-amplification in situ
hybridization does not have to be carried out. An
alternative method of visualization has been reported by
Embleton et al. using fluorochrome-conjugated primers
and subsequent analysis of positive cells by fluorescence
activated cell sorting (FACS) analysis. l 4 To date, there is
no study which has specifically compared the various
reported protocols. We believe that this would greatly
assist those planning to use in situ PCR techniques.
ASSOCIATED PROBLEMS AND
TROUBLESHOOTING
Although there are now many publications detailing
the use of these techniques, most investigators would
admit that many problems still remain. Problems arise
with the performance and optimization of the technique
and, following amplification, in the interpretation of
results. As evidenced by the wide variety of techniques
suggested (see Table II), it seems clear that no univer-
sally applicable technique is currently available. The
type of starting material, the type of target, the fixation
conditions of the tissues, and other factors all appear to
influence the success of the reaction. It appears that a
protocol which works very well in one system does not
invariably work in a parallel model.
There are very few reports which describe the success-
ful use of the above techniques with paraffin wax-
embedded tissue sections. In general, in situ PCR does
not work well with paraffin wax-embedded material.’0” ’
PCR-ISH protocols sometimes will work, if the hot-start
modification is used and/or if multiple primer pairs are
utilized.’ The commonest problem encountered is reac-
tion failure. In most cases, this can be overcome by
checking the thermal conductivity from the block to the
glass slide to the tissue section using a thermocouple, or
by using a dedicated thermal cycler for use with in situ
PCR techniques and/or optimizing the concentration of
reagents used.
Undoubtedly, a major advantage of these techniques
is their sensitivity, although efficiency is compromised
with almost linear amplification achieved in most
systems. The use of freshly fixed cell preparations
appears to provide maximal amplification efficiency,
whilst archival material attached to glass slides in most
cases yields weak or negative results. SpeciJicity should
theoretically also be good. From the literature, it
appears that in situ PCR has less specificity and repro-
ducibility when compared with PCR ISH.lo-tlA prob-
lem is non-specific incorporation of nucleotide into
damaged DNA by Taq DNA polymerase, even at room
temperature. This phenomenon is DNA polymerase-
and cycle-dependent and can occur in the absence of
primers or even with hot-start modification. Recently,
Gosden et (11. have reported the use of strand break
joining in in situ PCR amplification carried out on
chromosomes to eliminate spurious incorporation.2o
Diffusion of PCR product out of the cells during ampli-
fication can lead to extracellular amplification and there-
fore, again. spurious results. Many authors have
suggested ways of minimizing this phenomenon, but the
easiest is to limit the number of rounds of amplification
during the PCR. Quantitation is not currently applicable
to the technique and papers that deal with this should
be viewed with caution. Table I11 presents possible
remedies to some of the aforementioned problems.
REACTION, TISSUE, AND DETECTION
CONTROLS
Like SPPCR, in situ PCR techniques also require
meticulous use of controls. All reaction set-ups require
reference control genes, known negative samples for the
target sequence, together with irrelevant primers and
irrelevant probes. Parallel SPPCR should be performed
for each assay.
Omission of Taq should produce a weaker signal than
when Taq is added. With in situ RT PCR, omission of
 IN SITU PCR 19

Table 111-Troubleshooting and improving the technique

Troubleshooting
* Cell detachment
* Evaporation of PCR solution
* Leakage of product
* Poor yield/failure
* Non-specific signals
- coat slides with APES or poly-L-lysine
- use a deep-welled slide
- seal amplification well
- increase the size of the product
- immunofixation with anti-DNA antibodies
- ‘hot-start’ PCR
- cold start with SSB, E. coli
- increase the concentration of Taq DNA polymerase
- increase the number of cycles
- check detection protocols

Improvements
* Minimizing fixation effects ~

microwave
(e.g., histone DNA cross-linking) - formamide ( f Klenow fragment)
- zinc formaldehyde

reverse transcriptase and RNase pretreatment should be
carried out with each assay in order to detect false-
positive results due to amplification of endogenous
DNA sequences. In addition, serial dilutions of positive
cells provide an internal quantitative control. Post-
amplification immunohistochemistry has been described
by NUOVO,~ but most investigators find this difficult
to perform, because many epitopes do not appear to
withstand the repetitive changes of thermal cycling.
THE FUTURE OF ‘IN SZTU PCR’
Although in situ PCR techniques largely remain
research tools, their potential for practical diagnostic
pathology is Currently, PCR ISH and in
situ PCR have little to offer over conventional in situ
hybridization. In the future, however, the techniques
may be useful for the detection of low copy number viral
infections, in the study of the cellular expression of
oncogenes and growth factors, and in the diagnosis of
chromosomal translocations in certain tumour types,
including lymphomas, PNETs, and Ewing’s sarcomas.
The combination of RT in situ PCR with flow cytometry
also offers enormous potential for the future. Against
this, however, is the reality that the technique is still
capricious and PCR plus NISH techniques are inher-
ently complex and expensive. Standardization of instru-
mentation and simplification of the technique are
required in the near future if it is to receive universal
acceptance.
Should all pathology departments invest in the tech-
nique and consider it for use in diagnostic pathology?
On the face of it, it would appear easy to set up in situ
PCR in laboratories already working with PCR and
NISH. It does not necessarily follow that in situ PCR
will work, since no universally applicable protocol is
available to cover all possible investigative permutations
and combinations. We would like to finish with a word
of warning for those about to embark on setting up
in situ PCR; cursory reading of the literature suggests
that these techniques are easy and straightforward to
perform, but this is not the case and extreme caution
should be exercised.
REFERENCES
1. Herrington CS, de Angelis M, Evans MF, Troncone G, McGee JO’D.
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