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Technical Advance |
From the Laboratory of Pathology,*
National Cancer
Institute, and the Clinical Pathology
Department,
Clinical Center, National
Institutes of Health, Bethesda, Maryland; and the Division of Cytokine
Biology,
Center for Biologics Evaluation and
Research, Food and Drug Administration, Washington, DC
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Laser capture microdissection (LCM) is a technology recently developed by our laboratory3-5 and commercialized by Arcturus Engineering (www.arctur.com). LCM enables the user to procure pure cells from stained heterogeneous tissue under direct high-power microscopic visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells (with intact DNA, RNA, and proteins) is retained and held on the transfer film. Direct visualization of the captured cells, with their histology intact, ensures that the correct population of cells is obtained.
In the present report we examine the sensitivity and precision of a complete procedure for conducting quantitative prostate-specific antigen (PSA) immunoassay6 of stained human prostate tissue cells selected and procured by LCM. PSA was chosen as the analyte prototype for technology development because of its importance as a prostate marker and the known cellular heterogeneity of expression of PSA by prostate immunohistochemistry.2 Precision analysis was conducted for different numbers of laser shots. Comparisons of the number of PSA molecules per cell were made between different stages of prostate cancer progression in the same prostate and compared with PSA immunohistochemistry scoring.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue was obtained following an Institutional Review Board-approved protocol from both the Urologic Oncology Branch of the National Cancer Institute (Bethesda, MD) and the Mayo Clinic (Rochester, MN). After surgery, the tissue samples were immediately flash frozen in liquid nitrogen. The tissue was then embedded in O.C.T. compound and stored at -80°C. Cases were selected based on the histology present in the tissue sections so that normal glands, prostate intraepithelial neoplasia (PIN), and adjacent carcinoma could be compared within the same patient. Prostate tissue cases were selected to include ample stroma to serve as a negative control. Lung tissue was used as a second negative control.
Sectioning and Staining
The O.C.T.-embedded tissue blocks were cut with a cryostat into 8-µm sections. After cutting, the sections were immediately placed on dry ice and stored at -80°C. Only one section was thawed and dissected at a time, to minimize degradation of proteins. Frozen sections on plain untreated glass slides were fixed with 70% ethanol for 10 seconds. After fixing, the slides were washed in deionized water; stained with Mayer’s hematoxylin for 30 seconds, followed by another water wash, bluing solution for 30 seconds, and a wash in 70% ethanol; and then stained with eosin for 90 seconds. The slide was then dehydrated with two 10-second washes in 95% ethanol and two 10-second washes with 100% ethanol. Finally, the slide was placed in xylene for 30 seconds.
Laser Capture Microdissection
The Arcturus PixCell II system incorporates an Olympus IX-50 microscope containing a microscope slide stage that is moved by a joystick.4 The operator uses the joystick to position the tissue under a fixed laser beam that can be focused from a 7.5-µm to a 30-µm diameter. The LCM transfer film is fixed to the undersurface of a vial cap (CapSure TM TF-100 transfer film carrier, 5-mm-diameter optical-grade transparent plastic; the matching vial is Brinkmann no. 22 36 430-8). The LCM cell procurement time was always less than 15 minutes. A 5.0–10.0-µl droplet of the extraction buffer was applied to the surface of the film containing the selected cells for 1 minute. The cap with the droplet on its undersurface was then inserted in the mouth of the matching vial containing 45 µl of the immunoassay dilution buffer. The sealed vial sample receptacle was frozen at -20°C and stored for less than 48 hours before assay. The thawed sample was held at 4°C for no longer than 2.5 hours before introduction into the immunoassay module.
Extraction Buffer
Buffer A was 1/1000 dilution of the following stock: 50 mmol/L Tris-HCl, 1% NP-40, 0.1% Na deoxycholate,150 mmol/L NaCl, 4 mmol/L EDTA, aprotinin (10 mg/ml), leupeptin (10 mg/ml), 10 mmol/L Na pyrophosphate, 2 mmol/L Na orthovanate, and 100 mmol/L phenylmethylsulfonyl fluoride. Buffer B was "T-Per" tissue protein extraction liquid reagent (obtained from Pierce; catalog no. 78510). Buffer C was 10 mmol/L Tris-HCl (pH 7.4), 0.1% Triton X-100,1.5 mmol/L EDTA, 10% glycerol.
PSA Immunoassay: Microparticle Enzyme Immunoassay
PSA was measured by an automated two-site chemiluminescent assay, using the ultrasensitive PSA reagent kit on the Immulite immunoassay analyzer called the Immulite Third Generation PSA assay6 (Diagnostic Products Corp., Los Angeles, CA). This assay is a solid-phase two-site sequential chemiluminescent immunometric assay. The solid phase, a polystyrene bead enclosed within a test vial, is coated with an antibody specific for PSA. The sample microdissected cell proteins solubilized in a buffer are introduced into the test unit and incubated for 30 minutes at 37°C. PSA in the sample binds to the surface of the bead. Unbound proteins are removed by washing. An alkaline phosphatase-labeled polyclonal goat anti-PSA antibody is introduced, and the test unit is incubated for another 30-minute cycle. After a further wash, the chemiluminescent substrate, a phosphate ester of adamantyl dioxetane, is introduced. The substrate undergoes hydrolysis, accompanied by the emission of light. The light is measured by a luminometer. Relative light units (RLUs) reflect the photons detected by the photomultiplier tube, which is proportional to the concentration of PSA in the sample.
Immunohistochemistry
Immunohistochemistry staining for PSA was conducted using an adaptation of the method as described by Fend et al.7 The frozen sections were desiccated and then fixed in acetone. After washing with 3% goat serum, the primary antibody (Dako A562) was incubated for 1 hour at room temperature. Avidin/biotin was used to label the secondary antibody, and peroxidase/diaminobenzidine was used as the substrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
describes the steps of
LCM cell procurement and protein solubilization. Figure 2
is an example of LCM cell procurement
of prostate epithelial cells. The full staining characteristics of the
procured cells are retained, allowing for the precise enumeration of
the number of cells transferred to the polymer cap film. The yield of
procured pure epithelial or carcinoma cells encompassed within a
standard 30-µm shot and the precision of cellular procurement were
tested by visually counting the number of cells removed from the tissue
and transferred to the film.
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Table 1
compares the number of laser
shots with the average total number of microdissected cells for a
series of example prostate cases. A standard laser spot diameter of 30
µm encompasses a mean number of five to seven cells. Individual
tissue cells can vary in their packing density, their shape, and their
volume. Consequently, the imprecision of the cell yield is greater with
fewer laser shots. The SD for the number of cells procured by one shot
was approximately 50% of the mean. In contrast, when 15, 25,or 100
shots were procured, the SD of cell yield was reduced progressively to
less than 10% of the mean.
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After placement of a 5-µl microvolume of extraction buffer on the transfer surface containing the tissue cells, visual confirmation was obtained, establishing the complete solubilization of the stained cells. This took less than 1 minute. The transfer cap with the extracted cellular proteins was inserted into the mouth of a vial containing the immunoassay buffer. After analysis, the transfer caps were counterstained to ensure complete solubilization of the cells. The extraction buffer diluted in the assay buffer did not interfere with the immunoassay background level or linear calibration. Buffers A and B (described in Materials and Methods) were found to be highly suitable for solubilization and extraction of the PSA cytoplasmic protein from cells. Buffer C was determined to be suitable for membrane proteins. Her-2-Neu, a membrane-bound receptor, was solubilized in Buffer C from LCM-microdissected breast carcinoma cells and successfully measured using the Oncogene Science Diagnostics HER2/neu enzyme-linked immunosorbent assay (data not shown).
Calibration Curve
Negative tissues or zero controls produced a value of 80,000 to
140,000 RLUs, compared to PSA positive samples that ranged from 600,000
to 10,000,000 RLUs. The calibration curve used for measurement of PSA
in serum according to the Immulite package insert was applied to the
measurement of cellular tissue proteins procured by LCM. A specific
calibration curve for LCM-procured samples was developed with the
LCM-extracted cellular proteins in the buffer solution. As shown in
Figure 3, A and B
, the standard curve
using spiked known concentrations of PSA showed a high degree of
linearity (R2 = 0.98) over a
wide dynamic range (0.002–10 ng/ml). A strong degree of linearity was
also observed between the number of laser shots independently procured
in a case sample and the number of PSA molecules assayed (Figure 3C
and
Table 2
). A parallel study was
conducted on nine microdissected case samples. A representative example
series is shown in Table 3
. The assay was
conducted in duplicate on both undiluted and diluted (10 serial)
samples, and the observed and expected values were compared. For all
nine case samples the linear correlation between the observed and the
expected values was greater than R2
=
0.95. Thus the LCM-Immulite PSA immunoassay maintains good linearity
over the calibration range.
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The detection limit of the assay, defined as the concentration two
standard deviations above the signal response of a tissue extraction
sample free of PSA (zero dose) (Table 2)
, was found to be 0.002 ng/ml.
The "functional sensitivity," defined as the lowest concentration
measured with an interassay CV of 20%, was 0.005 ng/ml of PSA, or
approximately 4.5 x 106
molecules of PSA
per assay, using an extraction buffer microvolume of 5 µl. Depending
on the relative concentration of PSA expressed in the tissue cells
microdissected, in some cases the number of PSA molecules encompassed
in an individual shot (average of five to seven cells per shot) was
above the detection limit threshold.
Within-Run Precision
The LCM-Immulite assay precision is shown in Table 1
and Figure 4
. In keeping with the assumed biological
heterogeneity, one laser shot exhibits a PSA measurement CV of 60.2%
(n = 10), even though the mean magnitude of the
signal (804,416 RLUs) is substantially greater than the mean value for
100 combined shots of background tissue (132,759). Thus, capturing 15
(analytical sensitivity threshold) to 100 laser shots of a tissue
sample (this takes less than 5 minutes) provides an acceptable level of
precision for a routine assay. A time course study determined (in the
absence of protease inhibitors) that tissue proteins retained
immunoreactivity for a total microdissection time of at least 30
minutes (data not shown).
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Twenty cases were randomly chosen to include 10 normal and 10
tumor cases. Based on the results of the precision studies (Tables 1 and 2)
, a sample of 50 shots or greater has acceptable precision but
retains adequate cellular purity. Therefore, 10 replicates of 50 shots
were completed for each case. The average number of PSA molecules per
cell per sample and the SD of 10 within-run replicates are plotted in
Figure 4
. All 10 normal (epithelium) cases exhibited a PSA
concentration per cell of less than 2,500,000 molecules. The SD (10
replicates of 50 shots) of every case (for both normal and cancer
cases) was approximately 10% of the mean or less. In contrast, the
range of values for the cancer cases was up to sevenfold higher than
the normal cases, whereas the average of all of the invasive carcinoma
cases was 2,854,141 molecules. Nevertheless some tumor cases had values
within or below the mean value for the normal cases. This finding
validates at a molecular level, in tissue, the expected biological
heterogeneity and the phenotypic instability of carcinoma cells
compared to their normal counterparts.
Correlation with Tissue Immunohistochemistry
Analysis using LCM coupled with PSA immunoassays for PSA was
conducted on prostate tissue sections that contained normal epithelium,
carcinoma, and prostate intraepithelial neoplasia (PIN). Based on the
precision analysis described above, the number of laser shots was 100.
For each specimen studied, a dilution series such as that shown in
Table 3
, corresponding to a series of different numbers of laser shots,
was prepared. This verified that conclusions based on 100 shots
remained in the linear portion of the curve (Figure 3)
.
Immunohistochemistry for PSA was conducted6
on adjacent
sections. The PSA immunoreactivity of cellular populations identical to
those sampled by LCM on matched coded sections was scored independently
and ranked on a scale of 0 to 3+ (Table 4)
. The average number of PSA
molecules measured per cell ranged from 104
to
106. The immunohistochemistry scoring values
paralleled the quantitation and mirrored the heterogeneity in PSA
production by normal and neoplastic cell populations. Case C is an
example in which the normal epithelium contained an unusually high
level of PSA molecules (6.3 x 106) and was
scored 3+ by immunohistochemistry. In contrast, the PIN cells contained
3.7 x 105
molecules and was scored 2+, and
the tumor cells contained 1.99 x 104
and
stained 1+, all in the same case (case C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous methods for extracting and analyzing protein macromolecules from tissue subpopulations have included UV laser ablation of unwanted tissue regions10 and oil well isolation of tissue cells.8 These methods were complicated and labor intensive, did not use protein stabilizers, and did not employ soluble immunoassays. The advent of LCM now provides a means of positively selecting the desired tissue cells, even if they are scattered or in a spatially complicated distribution. Moreover, the protein macromolecules of LCM-procured cells appear to be intact and retain their functional activity.3 An important principle of the current methodology is exposure of the microdissected cells captured on the transfer surface to a microvolume of extraction buffer. The extracted molecules are directly diluted in the immunoassay buffer. In this way the microdissected cells are exposed to adequate local concentrations of detergents and other extraction chemistries that may interfere with antibody binding reactions. After dilution in the assay buffer, the extraction chemistry has no discernible residual effect on the immunoassay performance. The procurement and extraction procedure used in the present report did not alter the tissue cellular proteins in relative abundance, molecular weight, or charge, as judged by 2-D gel electrophoresis of proteins extracted from frozen sections compared before and after LCM procurement and staining of 7500 shots (data not shown). 2-D gel analysis of microdissected prostate tissue cells revealed PSA to be of low to moderate abundance compared to other cellular proteins. The current study was performed using frozen tissue stained with H&E. Fixed tissue embedded in paraffin is superior to frozen sections at the level of microscopic histomorphological details. Formalin fixation may produce cross-links that hamper protein extraction. In contrast, we have found that ethanol-fixed,7 paraffin-embedded tissue retains morphological details and preserves PSA immunoreactivity after microvolume extraction.
The present technology provides one of the first direct estimates of
the actual number of protein molecules per tissue cell in
vivo, for a single specific known protein of moderate to low
abundance. The number of total PSA molecules in normal prostate
epithelium ranged from 104
to
106
per cell. PSA is an important serum analyte
used to clinically monitor prostate cancer, but it is not a specific
marker of prostate cancer.11
Previous investigators have
reported great heterogeneity in the intensity of PSA
immunohistochemical staining among various neoplastic and nonneoplastic
populations in the prostate.2,12-16
Populations of
microdissected cells of a pure histological class (normal epithelium,
PIN, or invasive cancer) would be expected to contain some level of
heterogeneity in PSA expression among the population members. Sources
of imprecision and assay variability should therefore include the
following components: 1) precision in the number of cells captured per
sample, 2) normal biological variation in the production of PSA by
different cells from the same tissue and the position of the cells in
the plane of the tissue section,2,12-16
and 3)
imprecision in the immunoassay itself. Comparing the precision of cell
capture (Table 1)
with the variability of PSA quantity measured (Table 2)
, the percentage coefficient of variation (CV n = 10)
declines with increasing number of laser shots per sample. Acceptable
precision can be obtained with a sample of 50–100 laser shots. This
number of shots takes less than 5 minutes of operator time.
PSA immunolocalization in carcinoma cells is known to be increased or
decreased in intensity compared with normal epithelium in the same
prostate. Tissue cells positive for cytoplasmic PSA immunoreactivity
contain antigen-positive material in the endoplasmic reticulum and
secretory vesicles by immunoelectron microscopy and express PSA mRNA by
in situ hybridization. Application of the new technology
described in the present report (Table 4)
provides quantitative
confirmation of the heterogeneity in PSA expression that was previously
detected only by qualitative staining. The average numbers of PSA
molecules harvested per cell ranged over several logs. An
immunohistochemical staining difference could be discriminated within a
five- to 10-fold difference in PSA molecule number per cell. The level
of variability in PSA molecules per case is further validated by the
replicates of normal and invasive carcinoma cases shown in Figure 4
.
Here the normal cases were relatively homogeneous in the level of PSA
expression, whereas the tumor cases varied considerably.
In conclusion, the combination of LCM with high sensitivity chemiluminescence immunoassay chemistry provides a protein quantitation technology that can be applied to the measurement of proteins in microscopic pure populations of stained tissue cells. The speed, sensitivity, and linearity of the assay are acceptable for routine use, even considering the expected biological heterogeneity of histologically similar cell types. LCM immunoassays can thereby supplement or complement immunohistochemistry. Several extraction buffers were found to be suitable for cytoplasmic or membrane proteins (Materials and Methods). In addition to PSA, described here, the technology has been successfully been applied to the measurement of HER-2/neu assays (data not shown). Moreover, solubilized proteins can be analyzed by nonimmunological means (eg, enzymatic activity). With the application of microfluidics, and the multiplexing of assays, it is foreseeable that entire panels of quantitative protein assays can be performed on microscopic regions of pure tissue cells. This will provide the closest possible snapshot of the in vivo state of the protein pathways in developing, diseased, or genetically altered tissue.
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Footnotes |
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Accepted for publication October 27, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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