(American Journal of Pathology. 2001;158:1985-1990.)
© 2001 American Society for Investigative Pathology
A New Method for Histological Microdissection Utilizing an Ultrasonically Oscillating Needle
Demonstrated by Differential mRNA Expression in Human Lung Carcinoma Tissue
Michael Harsch*,
Klaus Bendrat*,
Gerhard Hofmeier
,
Detlef Branscheid
and
Axel Niendorf*
From the MEDEEA Forschungs-GmbH,*
Hamburg; the
Eppendorf Instrumente GmbH,
Hamburg; and the
Department of Thorax Surgery,
Hospital
Grosshansdorf, Grosshansdorf, Germany
 |
Abstract
|
|---|
Molecular analysis of microdissected tissue samples is used for
analyzing tissue heterogeneity of histological specimens. We have
developed a rapid one-step microdissection technique, which was
applied for the selective procurement of tissue areas down to a minimum
of 10 cell profiles. The special features of our microdissection system
consist of an ultrasonically oscillating needle and a piezo-driven
micropipette. The validity of this technique is demonstrated in human
lung large-cell carcinoma by real-time quantitative reverse
transcriptase-polymerase chain reaction assays of vimentin,
cyclin D1, and carcinoembryonic antigen after linear RNA
amplification. mRNA expression values of microdissected samples
scattered around those of bulk tumor tissue and showed differential
mRNA expression between samples of tumor parenchyma and supportive
stromal cells for vimentin and carcinoembryonic antigen as confirmed by
immunohistochemistry. In conclusion, this procedure requires
simple equipment, is easily performed, and delivers
microdissected tissue samples of oligocellular clusters suitable for
further molecular analysis.
|
Introduction
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The study of molecular genetic alterations that occur in tumors
requires a precise correlation of molecular genetic characteristics to
defined cell populations. In addition to the heterogeneity of the
neoplastic cell, tumor tissues are composed of a variable admixture of
stromal cells, inflammatory infiltrates, endothelial cells, and
pre-existing tissue. The presence of multiple cell types may dilute out
the significant changes that occur in specific cells and therefore most
sophisticated methods in molecular pathology are of limited value when
applied to bulk tissue.1
Several mechanical techniques for
microdissection have been developed to isolate cells for analysis from
histological sections. These include starch-based adhesive
fractionation,2
the use of
scalpel blades,3
fine needles,4-6
and
pipettes,7
either hand-held or connected to
micromanipulators. In an alternative method, isolation involves the
destruction of unwanted genetic material by UV irradiation after ink
protection of selected cells.8
Although these methods
achieve good precision they are time-consuming and laborious. UV lasers
are used to ablate unwanted tissue or to cut around selected cell(s) to
avoid contamination from adjacent tissue. Cells thus isolated were then
retrieved with needles (laser-assisted cell picking)9-11
or catapulted with slightly defocused laser shots directly into caps of
PCR tubes to procure cells in a noncontact manner (laser pressure
catapulting, LPC).12
Membrane-mounted tissue facilitates
removal of selected cells for either needle transfer or
LPC.12,13
In laser capture microdissection
(LCM)14
an infrared laser focally melts a transfer film,
which fuses to underlying selected cells. Both, LCM and LPC allow an
especially rapid dissection and are easy to perform; however, the
equipment required for laser-assisted methods is very expensive. To
overcome these drawbacks of current microdissection methods, we
developed a new mechanical technique, using an ultrasonically
oscillating needle and a piezo-driven micropipette for rapid one-step
histological microdissection. To validate our technique parenchymal and
stromal cells of human lung large-cell carcinomas were quantitatively
analyzed for differential mRNA expression of the intermediate filament
vimentin, carcinoembryonic antigen (CEA), and the
proliferation-associated antigen cyclin D1 and the results were
compared to their immunohistochemical distribution. We used an
analytical protocol that allows quantitation of multiple transcripts
from a single microdissected sample as small as 10 cells, using linear
amplification of RNA followed by real-time reverse
transcriptase-polymerase chain reaction (RT-PCR).
|
Materials and Methods
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Tissue Preparation
Parallel blocks of fresh tumor tissues were fixed in 4% buffered
formaldehyde and embedded in paraffin or flash-frozen in liquid
nitrogen and stored at -80°C until further analysis. Paraffin
sections were immunostained for vimentin (monoclonal antibody M7072,
1:400 dilution; DAKO, Hamburg, Germany) and CEA (monoclonal antibody
M7020, 1:100 dilution; DAKO) using the DAKO ChemMate Detektionskit
(DAKO) according to the manufacturer’s protocol.
For microdissection, 10-µm sections were cut from frozen blocks on a
standard cryostat and mounted on plain glass slides. Sections were
immediately hematoxylin and eosin-stained and dehydrated in graded
alcohols and xylene (10 seconds each). All solutions were prepared with
diethyl pyrocarbonate-treated H2O. To improve RNA
stability, slides of uncovered cryosections were stored at 4°C on
silica gel in an exsiccator.
Microdissection
Microdissection was performed using the prototype of a new
commercial microdissection device (MicroDissector; Eppendorf AG,
Hamburg, Germany). The system consists of a cutting head, a
micropipette, and an electronics box to control them. Both tools
(weight <50 g) were mounted on joystick-controlled three-axis
motorized micromanipulators (TransferMan, Eppendorf AG) that were
attached to an inverted microscope (Axiovert 35M; Carl Zeiss,
Oberkochen, Germany) (Figure 1)
. In the
cutting head longitudinal vibrations of an electrolytically sharpened
steel needle (tip radius, <0.5 µm) are induced by a monolithic
low-voltage piezoelectric actuator (5 x 2 x 5
mm3; CeramTech, Lauf, Germany). The cutting head
is machined from a solid piece of aluminum, where the vibrating beam
taking up the needle is linked to the body by two parallel members via
flexure hinges. The micropipette works with two normally convex brass
membranes (diameter, 35 mm) with glued-on piezo ceramic disks mounted
face to face (Piezosignalgeber-Membran EPZ-35; Bürklin, Munich,
Germany). With a DC voltage from between -30 and +250 V the membranes
flatten, thus effecting an arbitrarily variable displacement of up to
30 µl.
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Figure 1. Microdissection device. Inset: Schematic illustration of the
one-step preparation process. By use of the oscillating needle the
tissue for analysis is fragmented into subcellular particles that are
aspirated into the pipette tip.
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For microdissection the cryosections were covered with a pool of 15
µl of xylene. Under excellent visualization, areas of interest could
be easily dissected while moving the ultrasonically oscillating needle
in a meander-formed course through the selected tissue area (Figure 2)
. Along the path of the needle a
10-µm wide gap (Figure 2
, insert) was developed where the tissue was
detached from the glass slide and fragmented into subcellular
particles. The appropriate settings of frequency and amplitude (25 to
55 kHz and 0 to 2 µm) were dependent on the resonance of the needle
and were adjusted by observing the interaction of the needle with the
tissue. The micropipette, equipped with a GELoader Tip (Eppendorf AG),
enabled a continuous aspiration of the generated tissue particles with
xylene. Following this protocol, preparation and removal of tissue to
be microdissected could be done simultaneously in a one-step procedure
(Figure 1
, insert). The pipette tip was positioned close to the area to
be dissected, with a distance between needle and tip of 10 to 20 µm
at the beginning of dissection. There is no necessity to move both
together during preparation. A distance up to 400 µm is acceptable
when larger tissue areas were prepared. Tissue damage was avoided by
the elasticity of the pipette tip. The inner diameter of the pipette
tip, 0.15 mm, resulted in a high velocity of xylene flow dragging
tissue particles into the tip. The rate of xylene aspiration was
regulated at the control unit and adjusted to the speed of dissection.
Normally, a total volume of 
15 µl was continuously aspirated in
<2 minutes allowing the dissection of up to 1000 cells. The process of
dissection was paused and restarted by controlling the ultrasonic
actuation of the needle as well as suction with the micropipette by
foot pedals. This was particularly useful for collecting multiple
disseminated tumor-cell clusters. For subsequent analysis the xylene,
containing generated tissue particles, was transferred into a
microcentrifuge tube using the micropipette in its ejection mode.
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Figure 2. Photomicrographs of a human large-cell lung carcinoma showing tumor
parenchyma cell clusters during dissection using the oscillating
needle. The generated tissue particles were aspirated into the pipette
tip (not shown).
Inset: Cutting line in tumor stroma developed along the path
of the oscillating needle. Xylene-covered 10-µm cryosection. H&E
stained; original magnifications: x100, x200
(inset).
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RT-PCR results of samples prepared with the oscillating needle were
compared to two tumor parenchyma cell clusters that were microdissected
as single fragments according to Going and Lamb4
with the
modification that the tissue was covered with lysis buffer (PUREscript
RNA Isolation Kit; BIOzym Diagnostik, Hess., Oldendorf, Germany)
instead of proteinase K buffer solution.
Isolation of Total RNA and RNA Amplification from Microdissected
Samples
Total RNA of the cell clusters microdissected as single fragments
were extracted with the PUREscript RNA isolation kit (BIOzym
Diagnostik) and for tissue particles generated with the oscillating
needle with the Micro RNA isolation kit (Stratagene, Heidelberg,
Germany) after evaporation of xylene in a vacuum concentrator
(concentrator 5301; Eppendorf AG, Germany) according to the
manufacturers’ protocols. Total RNA was dissolved in 5 µl of diethyl
pyrocarbonate-treated H2O and digested 15 minutes
with 5 U RNase-free DNase I (Roche Molecular Biochemicals, Mannheim,
Germany) and 20 U RNase inhibitor (Life Technologies GmbH, Karlsruhe,
Germany) followed by incubation at 95°C for 5 minutes. Control PCRs
for genomic contamination were always negative.
The protocol for cDNA synthesis using an oligo-dT primer with an
additional T7 promoter sequence and subsequent RNA amplification has
been previously described in detail [Barry C, Pat Brown Lab:
Modified Eberwine "antisense" RNA amplification protocol
(http://cmgm.stanford. edu/pbrown/protocols/ampprotocol_2.txt)].
The only modifications were: 1) after double strand cDNA (ds cDNA)
synthesis the alkaline digestion of cellular RNA was omitted; 2) before
in vitro transcription (ds cDNA) was washed three times with
200 µl of diethyl pyrocarbonate-treated H2O
using a Microcon YM 100 centrifugal filter (Millipore GmbH, Eschborn,
Germany); and 3) amplified RNA (aRNA) was precipitated after organic
extraction with an equal volume of isopropanol in the presence of 20
µg of glycogen carrier. The aRNA pellet was washed with 70% ethanol
and dissolved in 4 µl of diethyl pyrocarbonate-treated
H2O.
RT-PCR from Microdissected Samples
First-strand cDNA was prepared from aRNA using the Expand Reverse
transcriptase kit (Roche Molecular Biochemicals) and random hexamers
according to the manufacturer’s protocol. The cDNA was diluted 1:2
(40-µl final volume), and aliquots of 1 µl were analyzed by
real-time PCR with a LightCycler instrument15
using the
primer pairs given in Table 1
. The
reaction was set up with the LightCycler-FastStart DNA Master SYBR
Green I (Roche Molecular Biochemicals) according to the manufacturer’s
protocol containing 1 µmol/L of each primer and a
Mg2+ concentration of 2.25 mmol/L. Amplification
conditions were modified to 95°C for 5 minutes, followed by 55 cycles
of 95°C for 15 seconds, 60°C for 5 seconds, and 72°C for 12
seconds with a temperature transition rate of 5°C/second from
annealing to extension. PCR products were identified by melting curve
analysis and by agarose electrophoresis (Table 1)
.
RT-PCR Analysis from Bulk Tumor Tissue
Isolation of total RNA from frozen tumor blocks (High Pure RNA
tissue kit, Roche Molecular Biochemicals) and oligo-dT primed cDNA
synthesis (Expand Reverse transcriptase kit, Roche Molecular
Biochemicals) were prepared according to the manufacturers’ protocols.
Real-time PCR analysis was performed as described above.
Data Analysis
Expression data were calculated using the LightCycler software.
PCR product accumulation was determined by measuring the fluorescence
once per cycle at the end of the extension phase to generate an
amplification curve for each sample. The cycle number at the
intersection of the log-linear region of the amplification curve with a
threshold of constant fluorescence for each sample was used to
calculate the expression data relative to a standard. Individual
standard dilution series were analyzed for each transcript where the
standard curve is a plot of log dilution factors to corresponding cycle
numbers. Expression of target genes was then normalized to ß-actin
expression to correct for losses during sample preparation and the
different cell amount in samples.
Quantitation of low-copy number transcripts is hampered by the
appearance of unspecific PCR products, which can be discriminated from
specific product by melting curve analysis, because of the nonselective
ds DNA binding of the SYBR Green I dye. Unspecific PCR products were
only detected in samples found negative for the analyzed transcript.
Statistical analysis was performed by use of SigmaStat for Windows
(Jandel). Data that passed the appropriate constraints of equivalent
variances and normal distribution were analyzed with unpaired
Student’s t-test, whereas other data were analyzed by the
Mann-Whitney rank-sum test. P values <0.05 (two-tailed)
were considered indicative of a statistically significant difference.
Data are presented as means ± SE.
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Results
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The use of the oscillating needle allows a micrometrically precise
dissection of cells for analysis and their easy detachment from the
glass slide. Even in rigid tumor stroma the oscillating needle retains
its excellent cutting ability (Figure 2)
. The time required for sample
preparation varied depending on the procured cell number and tissue
architecture. Small tumor-cell clusters were dissected in 20 seconds
and areas of 5000 cells were dissected in 6 minutes. During
preparation of larger areas the xylene pool became enriched with the
tissue particles. Remaining tissue particles can be removed under
visual control by repeated covering and aspiration with xylene and
pooled together with the initial sample. Negative controls (xylene,
aspirated from the section) before and after microdissection were
collected and subsequent RT-PCR after linear amplification revealed a
level of ß-actin in a ratio of 0.1% compared to a 200-cell sample.
Controls for genomic DNA contamination failed to generate a detectable
signal for a 110-bp fragment of the ß-globin gene (data not shown).
Our method was tested for the selective procurement of 5000 down to a
minimum of 10 cell profiles. RT-PCR results are shown in Figure 3
. Except for CEA, mRNA expression values
of microdissected samples scattered around those of bulk tumor tissue.
In samples of parenchyma vimentin mRNA expression was lower in both
tumors, CEA mRNA expression was higher in tumor B, and cyclin D1 mRNA
expression was higher in tumor A, when compared to stroma samples
(statistical analysis is given in legend Figure 3
). mRNA expression
values of the two samples (3k), which were microdissected as single
fragments were in the range of those prepared with the ultrasonically
oscillating needle. The threshold cycle of ß-actin was 18.79 ±
1.33 for 5000 cells (n = 5), 23.35 ± 1.06
for 500 cells (n = 8), and 27.82/27.39 (tumor
A/B, respectively) for 50 cells.
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Figure 3. Results of RT-PCR analysis. Each symbol represents expression values of
microdissected samples (red circle,
tumor parenchyma A, n = 6; green circle,
tumor stroma A, n = 4; red square, tumor
parenchyma B, n = 5; green square, tumor
stroma B, n = 4). The prepared
cell number is given in each symbol (k =
x1000, c = x100). Samples with indicated
cell number of 3k were microdissected as single fragments
(see Materials and
Methods). Lines as indicated
represent expression values for bulk tissue of tumors A and B.
Expression values are given normalized to ß-actin mRNA. Note:
expression values cannot be compared between different transcripts
(see Data Analysis). 0,
PCR product of target gene was not detected. Expression values of
microdissected samples were different between tumor parenchyma and
stroma for: vimentin 0.15 ± 0.003 versus 0.25 ±
0.078** (tumor A) and
0.04 ± 0.013 versus 0.13 ± 0.042*
(tumor B), CEA 5.9
± 4.2 versus 0.0002 ± 0.0002**
(tumor B), and cyclin D1
2.6 ± 0.64 versus 0.2 ± 0.06**
(tumor A). *, Student’s
t-test; **, Mann-Whitney rank-sum test.
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Immunohistochemical staining for vimentin showed similar patterns
of distribution in tumors A and B, revealing a positive stromal
reactivity and detection of a few scattered cells in parenchyma (Figure 4A)
. In tumor B >70% of parenchyma
cells showed immunohistochemical staining for CEA, whereas parenchyma
of tumor A and the stroma of both tumors remained negative (Figure 4B)
.
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Figure 4. Photomicrographs of immunohistochemical staining
(brown) of human
large-cell lung carcinoma for vimentin, showing the typical detection
of tumor stroma and scattered cells in tumor parenchyma
(A) and
CEA-positive parenchyma of tumor B
(B). Paraffin
sections; original magnification, x400.
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Discussion
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Analysis of pure cell populations is a prerequisite in the study
of differential gene expression in complex tissues, especially with
regard to advanced molecular techniques. As microdissection comes into
widespread use there is an increasing need for an economical method to
be applicable in routine work. In the present report we describe a new
mechanical technique using an ultrasonically oscillating needle and a
piezo-driven micropipette for rapid and precise histological
microdissection. The main advantage of our method over current
mechanical approaches is the improved speed of dissection and the easy
detachment of tissue from the slide. The method can be applied to
either paraffin sections or unfixed cryostat sections that are often
recommended for mRNA extraction. This can be an advantage because
compromised LCM after short drying periods of frozen sections has been
reported.16
The preparation with the oscillating needle
allows a sharp demarcation between the dissected area and unwanted
tissue that remain intact for further analysis. An additional example
of the efficiency of our method is demonstrated by dissecting
individual colonic crypts without collecting any adjacent stroma
(Figure 5)
.
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Figure 5. Photomicrograph of colonic mucosa showing completed microdissection of
single colonic crypts. The surrounding connective tissue remained
entirely intact. Paraffin section; original magnification, x200.
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The most sophisticated and certainly the best techniques
are laser-assisted methods suitable for microdissection of single cells
with minimized risk of contamination.12,17
However, our
technique competes with laser-assisted methods concerning
preparation time and precision for microdissection of oligocellular
clusters. For studies that focus on tissue samples in sizes as analyzed
in this report, our method offers an alternative at moderate cost
(estimated to be approximately $16,000 US dollars, excluding inverted
microscope and manipulators). The system is flexible and can be
assembled using a conventional inverted microscope with
micromanipulators that are frequently already present in the
laboratory. A manual micromanipulator has been tested and is adequate
for positioning the micropipette. The system is robust and
maintenance-free, running costs are limited to disposable pipette tips
and needles. In analogy to laser-assisted cell
picking9-11
where selected cells are isolated by
photolysis of adjacent tissue, our method should also be useful for the
microdissection of single cell profiles. Adjacent tissue can be removed
with the oscillating needle and micropipette and cells thus isolated
can be retrieved by conventional methods.
It is a general problem of microdissection that dry noncoverslipped
slides do not allow fine cytological detail. The preparation under
xylene provides excellent optical conditions, which can be particularly
helpful for the dissection of premalignant lesions.18
Furthermore, in contrast to aqueous buffer solutions, the tissue does
not suffer variable adherence to the glass slide, which can diminish
the precision of dissection4,5
and RNA stability is
improved in an anhydrous environment.
The validity of this technique is demonstrated by the RT-PCR data
(Figure 3)
. Quantitative analysis of mRNA for vimentin and CEA are in
concordance with immunohistochemistry, ie, differential mRNA expression
between tumor parenchyma and stroma was shown for these genes.
Additionally, differential mRNA expression between parenchyma and
stroma was shown for cyclin D1 in one tumor. mRNA for CEA always showed
highest expression in CEA immunohistochemically positive areas of tumor
B, however none of the expression values reached that of bulk tumor
tissue, indicating an inhomogeneous distribution of CEA expression in
tumor B. Microdissection of immunophenotypically defined cell
populations allows cell-specific mRNA analysis according to antigen
expression.10,16
Different amplification efficiencies for
CEA and ß-actin during linear amplification could also be a cause for
the lower relative expression of CEA in microdissected samples. Linear
amplification of RNA has been shown to be reproducible between
individual LCM dissected cells and was successfully used to obtain gene
expression profiles with cDNA microarrays.19
In our
protocol, linear amplification of polyA RNA and subsequent hot-start
PCR facilitated quantitative analysis of multiple transcripts from a
single sample, especially if only few cells are prepared or at low-copy
number transcripts. As assessed from further dilution steps of standard
series additional PCR cycles were critical for a precise quantitation,
because of the appearance of unspecific products.
In conclusion, we describe a new mechanical method for histological
microdissection. The use of an ultrasonically oscillating needle and a
piezo-driven micropipette were demonstrated as sufficient tools,
allowing a precise and efficient procurement of homogeneous tissue
samples. Our data support the utility of this technique for the
determination of gene expression in defined cell populations. The
moderate cost and low maintenance of the system will provide a
technique that could be accessible to a large number of scientists.
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Acknowledgements
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We thank Peter Gebhardt and Hartmut Schmidt-Rabenau for critical
discussions, Dieter Knofe for the graphics, Anke Nagel for technical
assistance, and Dr. David Evans for critically reading the manuscript.
|
Footnotes
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Address reprint requests to Prof. Dr. med. Axel Niendorf, Gemeinschaftspraxis für Pathologie, Lornsenstr. 4, 22767 Hamburg, Germany. E-mail: niendorf{at}uke.uni-hamburg.de
Accepted for publication March 2, 2001.
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Abstract
Introduction
