ARTICLE |
Correspondence to: Heike Allgayer, Dept. of Surgery, Klinikum Grosshadern, Ludwig-Maximilians Univ. of Munich, Marchioninistr. 15, 81377 Munich, Germany.
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Summary |
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Phenotyping of cytokeratin (CK)18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15-80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (106 bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1-280 in 106; double staining 808, range 0-253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant (p<0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients. (J Histochem Cytochem 45:203-212, 1997)
Key Words: immunogold/APAAP double staining, methodological aspects, uPA receptor, CK18-positive cells, bone marrow, phenotyping
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Introduction |
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Minimal residual tumor disease in solid epithelial cancers has been indicated during the last years by investigations of disseminated tumor cells in bone marrow identified by the marker cytokeratin 18 (CK18), a cytoskeletal component of simple epithelial and carcinoma-derived cells (
Our earlier results regarding the correlation of urokinase-type plasminogen activator receptor (uPA-R) on those cells, with their postoperative quantitative increase (
For reliable phenotyping of disseminated tumor cells, an immunocytochemical double staining method that can detect CK18-positive cells against the mesenchymal background of bone marrow with high sensitivity and reproducibility is necessary. The second antigen should be unequivocally identified as to its cellular localization, in good contrast to CK18 staining and without any crossreactivity.
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Materials and Methods |
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Patients
Bone marrow was taken from 219 patients who underwent surgery for gastric cancer. As a first step, APAAP staining was performed to screen those patients for CK18-positive cells in bone marrow. A total of 61 patients with positive CK18 results in APAAP and at least 106 bone marrow cells left for a second screening with our double staining method were involved in the present study.
Bone Marrow Aspirates
Bone marrow was taken intraoperatively from both iliac crests (5 ml each) and heparinized. Immediately after aspiration, the bone marrow underwent Ficoll-Hypaque density centrifugation (
Immunocytochemical APAAP Staining
APAAP staining for CK18-positive cells (
Immunocytochemical Double Staining
Biotinylation of MAb CK2 was done by dissolving 1 mg/ml CK2 in 25 µg D-biotinyl--aminocaproyl-N-hydroxysuccinimide (Boehringer) and 50 µl dimethyl-formamide (DMF), overnight incubation, and membrane ultrafiltration with a Centricon centrifugal microconcentrator (Amicon; Witten, Germany) to separate DMF and uncoupled biotin (
A modified double staining protocol based on the method described by ; American Diagnostica, Greenwich, CT) was incubated for 60 min, followed by gold-labeled goat anti-mouse antibody for 30 min (0.08 mg/ml, 1:50, Auroprobe One Reagent; Amersham, Braunschweig, Germany). To avoid crossreactions, 10% mouse serum/PBS (Dako) was applied for 25 min.
The second part of the double staining was performed using biotinylated CK2 MAb (10 µg/ml, 45 min) and AP-conjugated streptavidin (1.1 mg/ml, 1:100, 30 min; Jackson ImmunoResearch, West Grove, PA). In contrast to
Visualization of specifically bound CK2 was done with new fuchsin dye (0.40 mg/ml; Serva, Heidelberg, Germany), sodium nitrite (0.04 mg/ml; Merck, Darmstadt, Germany), levamisole (0.36 mg/ml; Sigma), 0.2 M Tris buffer (pH 8.7), and naphthol AS-BI phosphate (0.08 mg/ml; Sigma) dissolved in DMF.
After washing the slides thoroughly in bidistilled water, specifically bound 1-nm colloidal gold particles (uPA-R staining) were visualized by silver enhancement under microscopic control. Equal volumes of inducer and enhancer of a silver enhancement kit (Amersham) were mixed and immediately incubated at room temperature for a maximum of 40 min. The silver kit was completely exchanged after 20-min incubation to avoid unspecific precipitation of silver granules. Slides were washed in bidistilled water and mounted with Kaiser's glycerol gelatin (Merck).
For comparison of sensitivity between this method and the original protocol of
Slides of colon cancer cell lines SW403 and HT29 (ATCC; Rockville, MD) treated under the same conditions served as positive controls. For isotype crossreactivity controls, MAb CK2 was replaced by murine IgG1 (MOPC21; Sigma) and MAb against uPA-R by murine IgG2 (UPC 10; Sigma) in equimolar protein concentrations. Two other slides were stained without the first bridge and the second primary antibody, and vice versa. Two further slides underwent the staining procedure without the first second bridge. Bone marrow of a healthy donor served as another control.
Tumor Cell Dilutions for Determination of Sensitivity
The sensitivity of our method regarding detection of CK18-positive cells required testing in comparison to the original protocol (
Modification of the Double Staining Protocol
The original protocol of
The sensitivity increased to 1:100,000 with the omission of glutaraldehyde postfixation. This was not diminished in a second experiment with 10% mouse serum and omission of postfixation.
A final protocol was therefore established with 10 µg/ml CK2, 10% mouse serum, and omission of glutaraldehyde. Testing at logarithmic tumor cell dilutions comparing the modified protocol with the original was now done according to the preceding paragraph.
Investigation of uPA-R Expression in Normal Bone Marrow Cytospins
Expression of uPA-R in cultured normal bone marrow stimulated with cytokines has been recently described (
Analysis of Staining Results
All slides (including tumor cell dilutions) were coded and independently analyzed by two blinded investigators. Bone marrow preparations from patients were screened without knowledge of patient identity or stage of disease. Cell numbers detected by double staining were counted independently from single APAAP results.
Statistical Analysis
A correlation diagram (scatter plot) with calculation of the regression line and linear regression analysis (level of significance p<0.05) was applied for estimation of correlation between CK18-positive cell numbers detected in APAAP-Fast Blue and the double staining method, using the EDA statistical software package (Department of Medical Information, Biometry and Epidemiology, Klinikum Grosshadern, Munich, Germany). This program was also used for calculation of means, standard deviations (SD), and standard errors of the mean (SEM).
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Results |
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Staining Results and Controls
CK18-positive cells were easily detected by deep brownish-red staining of the cell cytoplasm, even at low magnifications (Figure 1 and Figure 3). Bone marrow from healthy donors was routinely stained as a negative control in each assay, and never exhibited CK18-positive cells.
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Expression of uPA-R on CK18-positive cells was indicated by black-grained linear staining of cell membranes (Figure 2). In some cases the staining appeared adjacent to nuclear membranes (Figure 4). Between 10 and 50 of 1000 surrounding bone marrow cells revealed specific staining for uPA-R. The specificity of this phenomenon was investigated in bone marrow from 10 donors with nonmalignant diagnoses (see below). UPA-R expression by CK18-positive cells was detected in 29 of the 61 patients investigated.
The tumor cell lines SW403 and HT29 showed almost 100% CK18-expression, with uPA-R expression between 15-80%, depending on the culture passage. A maximal percentage of uPA-R expression could be found at the beginning of tumor cell culture.
All isotype controls were negative, and neither specific nor unspecific red or black staining was detected.
UPA-R Expression in Normal Bone Marrow
To prove the specificity of uPA-R staining of bone marrow cells in our double staining method, we also stained bone marrow cytospin preparations from 10 patients with nonmalignant disease for uPA-R, using the established APAAP-Fast Blue protocol (
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Sensitivity of the Double Staining Protocol
The sensitivity of detection of expected CK18-positive cells was tested with tumor cell dilutions (SW403/PBL) described above (see Materials and Methods) using the original staining protocol of
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With a double staining protocol using 10 µg/ml biotinylated MAb CK2 and omitting the postfixation step with glutaraldehyde, we could optimize sensitivity up to 1 tumor cell in 300,000, with good visibility of stained cells. The results of this modified protocol compared to the original are shown in Table 2, which gives means and standard deviations of cell counts found in 10 slides with 105 bone marrow cells each. The results were confirmed by two replicates of the tumor cell dilution, one revealing no positive cells at dilutions of 1:100,000 and higher with the original protocol and 4 cells in 10 slides at 1:300,000 with the modified method, the second showing three hardly visible positive cells in 106 at 1:100,000 with the original (higher dilutions negative) and two clearly detectable cells at 1:300,000 with the modified protocol, with expected tumor cell numbers in all lower dilutions.
Estimation of Reproducibility of CK18-positive Cell Detection
Of our 61 patients, 1 million bone marrow cells (10 slides with 105 cells each) were stained with our modified double staining protocol and with the APAAP-Fast Blue method as well, as positive CK18 counts in the single APAAP method were defined as prerequisite for additional double staining. Therefore, comparison between quantitative CK18-positive results of both methods was expected to allow an estimation of the reproducibility of our double staining technique.
Detailed quantitative results of both methods are given in Figure 5 for each of the patients. It shows comparison of cells detected in APAAP (y-axis) with double staining (x-axis).
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In four cases (7%), our double staining method was unable to detect CK18-positive cells again. Therefore, the reproducibility of qualitative cell detection in APAAP by double staining was estimated as 93%.
In 56% of cases (n=34), double staining found fewer or equal cell numbers. In the four patients with zero redetection of cells by double staining, 7 cells had been found altogether with the APAAP technique (for the single cases range 1-3 cells in 106, mean 2.0, SD 0.8). In six further patients, APAAP detected 17 cells altogether (range 1-4 in 106 in the individual case, mean 2.8, SD 1.2), and the double staining method could redetect the same cell numbers again. In 24 patients, APAAP staining revealed 653 cells in total (range 2-280 in 106 per individual, mean 27.2, SD 54.2), and with 495 cells altogether double staining redetected fewer CK18-positive cells (range 1-253 in 106, mean 21.5, SD 48.9). In 44% of cases (n=27) double staining found more CK18-positive cells in bone marrow than did the APAAP-Fast Blue method. APAAP had revealed a total of 161 cells in these 27 patients (for each case range 1-40 in 106, mean 6.0, SD 8.0), and the double staining method found 296 cells (range 1-53 in 106, mean 11.0, SD 10.9). Overall, a relative quantitative CK18-positive cell detection of 97% (808 cells in total) compared to the APAAP method (838 cells in all 61 patients) was seen.
CK18-positive cell counts of the two methods were positively correlated (Figure 5) with r=0.76 and p< 0.001 (linear regression analysis).
Because variability of CK18-positive cell contents among cytospins potentially detectable by immunocytochemical methods should increase with a decrease in overall CK18-positive cell numbers in 106 and should be highest in cases with very low CK18-positive cell load, we additionally calculated the relative reproducibility of cell numbers for the 33 patients with 5 or more CK18-positive cells in single APAAP separately. Even in these cases with potentially more stable CK18-positive cell distribution, the double staining protocol revealed 94% (in total 735 compared to 783) of CK18-positive cells detected in single APAAP staining.
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Discussion |
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Our double staining method combines an immunoenzymatic step (streptavidin-biotin-APAAP) with an immunogold-method of high sensitivity.
The advantage of this combination compared to double enzymatic methods is avoidance of color mixing, which often leads to false-negative results and inability to clearly identify subcellular localizations of the two antigens investigated (
Compared to immunofluorescence, our method enables time-permanent preparations with visibility of colors for years, and is more cost-effective (
An alternative to immunocytochemical double labeling could be provided by flow cytometry, which also enables identification of more than one antigen even quantitatively by multiplex labeling at sensitivities reaching 1 cell in 1,000,000 (
The sensitivity of our double staining protocol was tested with logarithmic dilutions of tumor cells in comparison to Riesenberg's original protocol. It was seen that sensitivity (also considering easily visible staining intensity) could be improved from 1:10,000 to 1:300,000 by omitting postfixation with glutaraldehyde, because higher CK2 antibody concentration (10 µg/ml compared to 8 µg/ml in the original protocol) alone had resulted in only poor improvement of sensitivity (data not shown). The fixation mechanism of g lutaraldehyde is known to be alcylation of sulfhydryl and COOH groups of cell membrane proteins, which potentially decreases the permeability of cell membranes to different molecules (
Comparison of CK18-positive cell numbers detected with our double staining method with APAAP-Fast Blue results should allow estimation of reproducibility. In only four cases (CK18-positive cell numbers between 1 and 4; see Table 2) did our double staining protocol fail to reproduce positive cells. Summarizing cell numbers of the 61 patients investigated, our double staining protocol was able to detect 97% of CK18-positive cells in APAAP. In 44% of cases, the double staining method demonstrated even more cells than the APAAP method. This can certainly be explained by the variability of CK18-positive cell distribution within the cytospin preparations, especially in patients with low CK18-positive cell counts, as summarized from investigation of 10 cytospins with 105 bone marrow cells each. Leaving out those cases with very low CK18-positive cell counts in single APAAP (<5 in 106), we tried to decrease this influence, because with higher CK18-positive cell numbers the variability of detection due to incidental cell distribution between cytospins should become more stable. Even here, the double staining method detected 94% of the cell number in single APAAP, indicating good reproducibility. Nevertheless, it should be emphasized that this comparison is an approximation for the true reproducibility of our method, because different cytospins with potentially different CK18-positive cell numbers had to be stained with single APAAP and the double staining protocol.
Because the immunogold step of our method is known to be highly sensitive (
uPA-R marking was present not only on CK18-positive cells but also on bone marrow cells. False-positivity of this phenomenon was excluded by staining of normal bone marrow for uPA-R with the established APAAP method. Here, expression of uPA-R could be demonstrated for a mean of 6.5% of cytospin bone marrow cells. From the literature it is established that uPA-R is expressed on blood leukocytes (e.g., monocytes, neutrophilic granulocytes, activated T-cells (Kramer et al. 1994;
Other authors, using the same double staining method, also confirm the specific marking of the immunogold step.
The specificity of our method is further supported by permanently negative isotype and cross-matched controls. Background staining could be reduced to minimum by extensive washing between the incubation steps, the importance of which is also confirmed by
In our present investigation, uPA-R was mainly detected on cell membranes. In single cases of disseminated CK18-positive cells in bone marrow and also in tumor cell line-positive controls, staining for uPA-R was also localized on nuclear membranes. It is necessary in further investigations to verify this nuclear staining by electron microscopy. However, this finding is corroborated by the report of
In summary, our present investigation suggests a double staining protocol that allows highly sensitive characterization of disseminated CK18-positive cells in bone marrow, as well as good reproducibility, by use of an established method. The first results regarding correlation of uPA-R detection on those cells with later increasing CK18-positive cell counts and a worse prognosis for cancer patients (
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Acknowledgments |
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We thank Boehringer Mannheim GmbH Research Center (Tutzing, Germany) and American Diagnostica (Greenwich, CT) for generously supplying antibodies.
Received for publication March 7, 1996; accepted September 18, 1996.
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