Virchows Archiv B Cell Pathol (1993) 64:67-73 9 Springer-Verlag1993

In-situ polymerase chain reaction

An overview of methods, applications and limitations of a new molecular technique

Paul Komminoth 1 and Aidan A. Long 2

1 Division of Cell and MolecularPathology, Department of Pathology,Universityof Ziirich, CH-8091 Ztirich, Switzerland
2 Department of Medicine,AllergyDivision, Tufts UniversitySchoolof Medicine;New England MedicalCenter Hospitals,
Boston MA, USA
ReceivedMarch 15 / AcceptedMarch 17, 1993

Summary. The in-situ polymerase chain reaction (in-situ ally limited by its detection sensitivity. Thus, threshold
PCR) is a novel molecular technique that combines the levels for messenger RNA (mRNA) detection are at
extreme sensitivity of the PCR with the anatomical local- about 10 to 20 copies per cell and conventional ISH
ization provided by in-situ hybridization. A number of techniques do not detect single copy DNA sequences.
groups have recently reported studies using in,situ PCR The polymerase chain reaction (PCR) is an extremely
for the detection of specifically amplified single-copy sensitive technique which can amplify rare or single copy
nucleic acid sequences in single cell preparations or low gene sequences to high levels, easily detectable by gel
copy DNA sequences in tissue sections. In this overview, electrophoresis and Southern blot hybridization. How-
we describe the principles of in-situ PCR, review the ever, nucleic acid extraction and cell destruction are gen-
applications of this technique and discuss future aspects erally required before the PCR amplification and, ac-
of in-situ PCR. We critically compare the different in- cordingly, neither the correlation of results with histo-
situ PCR protocols described in the literature. Emphasis pathological features, nor the enumeration of positive
is placed on the absolute requirement for controls to cells in a mixed population is possible.
allow accurate interpretation of results and the possible Recently, however, there have been several studies
problems and pitfalls of the in-situ PCR methods, in- describing in-situ PCR, a method that combines the high
cluding artefacts related to diffusion of PCR products sensitivity of the PCR with the cytological localization
and non-specific incorporation of labelled nucleotides of sequences provided by ISH. Such a technique could
into fragmented DNA undergoing repair. It is concluded have important diagnostic applications in infectious dis-
that this technique will eventually play an important ease and oncology.
role in specialized diagnostic laboratories in the evalua-
tion of viral diseases, haematological and other malig-
nancies which have unique genetic markers. Principles of in-situ P C R

Key words: In-situ polymerase chain reaction - In-situ In-situ PCR represents an attempt to wed the techniques
PCR - In-situ hybridization, ISH of PCR and ISH through the amplification of specific
nucleic acid sequences inside single cells and increasing
copy numbers to levels readily detectable by ISH or im-
munohistochemistry. The principles of in-situ PCR are
Introduction outlined in Fig. 1.
Cells or tissue samples are fixed and permeabilized
Certain clinical and research questions in pathology can- before in-situ PCR in order to both preserve morpholo-
not be answered by current immunohistochemical or gy and permit access of the PCR reagents to the intracel-
molecular techniques. One important limitation relates lular sequences to be amplified.
to the frequent inability to localize particular gene se- PCR amplification of target sequences is performed
quences to individual cells or cell types in mixed cell either in intact cells as a suspension in Eppendorf tubes
populations. While in-situ hybridization (ISH) does per- or directly in cytocentrifuge preparations or tissue sec-
mit localization of specific nucleic acid sequences at the tions on glass slides by overlaying the samples with the
level of individual cells and can do so with a very high PCR reaction mixture under a coverslip. Working with
degree of detection specificity, its usefulness is occasion- single cell preparations, fixed cells function as "amplifi-
cation sacks" with semi-permeable membranes, which
Correspondence to: P. Komminoth permit the primers, nucleotides and DNA polymerase
68

Principles of in-situ PCR a finding could not have been made by any other existing
technique. In viral diseases, this technique is useful not

@ Sample Fixation
for preservation of
morphology
only in the identification of cell types which actually
harbour a virus or provirus but may also permit esti-
mates of viral load in particular cells or tissues.
Two groups have also applied in-situ PCR to study
endogenous DNA sequences including human single
copy genes, rearranged cellular genes and chromosomal
Enzyme Primers translocations (Embleton et al. 1992; Komminoth et al.
Permcabilization 1992a; Long 1991; Long et al. 1993; Ray et al. 1991).
to allowintracellular Recently Embleton etal. (1992) successfully detected
penetrationof primers, amplified immunoglobulin mRNA sequences in single
nucleotidesandenzyme
Nucleofldes
cells, performing an intracellular reverse transcriptase
(RT) step prior to in-situ PCR ("in-situ RT-PCR"). By

1 exploiting the ability to amplify tumour specific nucleic

acid sequences as clonal markers of malignant cells (e.g.
unique immunoglobulin or T cell receptor gene rearran-
In-situ Amplification gements, chromosomal translocations or point muta-
by PCRto increasecopy tions in cellular proto-oncogenes), in-situ PCR has the
numbersof targetnucleic potential to facilitate accurate tumour diagnosis and per-
acidsequenceto detectable
levels mit quantitative estimations of residual disease after
therapy.

1
~ ~ I ! Visualization of PCR
Current achievements with in-situ PCR show that this
new molecular technique has the potential to advance
our understanding of many disease processes and may
products in-situ also provide important insights into the effects of specific
I - i n d i r e c t l y by in-situ
hybridization, therapies.
- d i r e c t l y by detection
of
labeled PCR products

Fig. 1. Principlesof in-situ PCR Comparisons of in-situ PCR protocols

Protocols for successful in-situ PCR have been indepen-

dently developed by several groups. A majority of these
to pass into the cell and nucleus, yet sufficiently retard protocols share important key characteristics (see
the outward diffusion of PCR products to allow their Fig. 1). Despite the conceptual simplicity of in-situ PCR
detection in-situ. Both block cyclers or cycling ovens as a general approach, important experimental differ-
have been used as equipment for thermal cycling. ences relating to sample preparation, in-situ amplifica-
Intracellular PCR products are then visualized either tion of target nucleic acids, detection of PCR products
by ISH (indirect in-situ PCR) or by direct detection of and use of controls exist. Details of technique, often
labelled nucleotides, which have been incorporated dur- unique to particular applications, emerge as crucial de-
ing PCR (direct in-situ PCR). terminants of eventual success (Long et al. 1992). The
variables encountered in different in-situ PCR protocols
are described in more detail below and are summarized
Applications of in-situ PCR schematically in Table 1.

To date, six groups have reported work with in-situ PCR Starting materials and equipment. The physical set-up
describing successful detection of specifically amplified employed for in-situ PCR with cellular material has var-
single copy nucleic acid sequences in single cells and ied among different groups. In-situ PCR in single cells,
low copy DNA sequences in tissue sections. Most of first described by Haase et al. (1990), used fixed cells
the studies have focused on the detection of viral (for- suspended in the PCR reaction mixture in Eppendorf
eign) DNA sequences (e.g. Visna-virus, HIV, HPV, tubes, which were thermal cycled in a block thermo-
MMTV provirus, CMV or HBV) in model systems (Ba- cycler. After PCR, cells were cytocentrifuged onto glass
gasra et al. 1992; Chiu et al. 1992; Embretson et al. slides for subsequent ISH visualization of intracellular
1993; Haase et al. 1990; Komminoth et al. 1992a; Long PCR products. Similar approaches have been used by
et al. 1993; Nuovo et al. 1991a, b; Spann et al. 1991; other groups (Embleton et al. 1992; Komminoth et al.
Staskus et al. 1991). A smaller number of studies have 1992a; Long etal. 1993; Ray et al. 1991). In one earlier
looked at clinical aspects of viral disease and recently study, pieces of glass slides with adherent cells from cyto-
made the surprising observation, that more cells and centrifuge preparations were incubated in the PCR mix-
cell types than previously suspected are latently infected ture in Eppendorf tubes (Spann et al. 1991). Of all the
by HIV during early and late stages of infection (Bagasra in-situ PCR protocols developed to date, it appears that
etal. 1992; Embretson etal. 1993; Kolata 1993). Such methods using intact cells suspended in PCR reaction