Feature Article
Novel Histochemical Stain for Tinctorial
Detection of Cancer and Neoplastic Cells
Pavel Idelevich1, Don Kristt2, Elimelech Okon3, Dov Terkieltaub1, Ilia Rivkin1, Ami Fishman4, Sylvia Lew5,
Eduardo Schejter6, and Adi Elkeles1
1
Zetiq Technologies Ltd., Ramat Gan, Israel
2
Molecular Pathology, Rabin Medical Center, Petah Tikva, Israel
3
LEM Pathology Laboratory, Nes Ziona, Israel
4
Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba, Israel, affiliated with
Sackler School of Medicine, Tel-Aviv University
5
Department of Pathology, Meir Medical Center, Kfar Saba, Israel
6
Women Health Center, Maccabi Health Services, Tel Aviv, Israel
Abstract
Zetiq has introduced a histochemical stain that claims to
tinctorially identify cancer and neoplastic cells. Because of
the potential importance of such a capability, we undertook
to investigate Zetiq’s CellDetect® staining technology as
applied to cultured cell lines as well as an initial sample of
clinical cases. This goal was pursued by investigating four
types of comparisons: 1) cancer cell lines before and after
differentiation; 2) cervical squamous-cell carcinoma (SCC)
biopsies to non-neoplastic squamous epithelium; 3) SCCs to
neoplastic, nonmalignant squamous epithelium; and 4) neoplastic squamous cells to non-neoplastic squamous cells in
cytological preparations. The clinical material was also
stained with hematoxylin and eosin (biopsies) or Pap (cytologies) for diagnostic purposes. We found that all CellDetect®stained cells exhibited one of the two tinctorial outcomes.
Cell lines with malignant phenotype uniformly had red/purple cytoplasm, whereas the differentiated phenotype changed
the color to blue/green. Moreover, these two color values are
sufficiently distinct that they can be readily distinguished
quantitatively with an ELISA reader when applied to cells in
tissue culture. Biopsies of SCC and non-neoplastic tissues
exhibit the same two color values: cancers had red/purple
cytoplasm, whereas non-neoplastic tissues were green/blue
stained. In contrast, neoplastic, nonmalignant tissues (dysplasias) stained red/purple, similar to SCCs. Cytological
preparations gave similar results: neoplastic cells stained
red/purple, whereas non-neoplastic cells exhibited green/
blue cytoplasm. The study demonstrated that the staining
was rapid and reproducible. Conclusion: The CellDetect®
stain allows detecting cancer and neoplastic cells tinctorially
Address reprint requests to Pavel Idelevich, Zetiq Technologies Ltd.,
5-7 Shoham St., Ramat Gan 52521, Israel. E-mail [email protected]
One continuing education contact hour can be earned by reading this
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The Journal of Histotechnology / Vol. 32, No. 3 / September 2009
based on a rapid, reproducible histochemical process. (The J
Histotechnol 32(3):97–105, 2009)
Submitted April 30, 2009; accepted with revisions August
13, 2009
Key words: cancer detection, cancer diagnosis, cell staining, cytopathology, histopathology, Warburg effect
Introduction
One productive area of oncologic research has focused on
improving methods for the pathological diagnosis of cancer.
Today, the core of cancer detection, diagnosis, and classification is still the light microscopic analysis of morphology
(1–4). Nonetheless, total reliance on purely morphological
criteria has limitations because the use of morphology alone
often fails to detect neoplasia (6), differentiate between different types of neoplasia, or distinguish reactive processes
from neoplasia. The latter issue often arises in regard to reactive mesothelial cells, which are present in serous effusions.
These cells may exhibit atypical features that are considered
characteristic of cancer cells, such as multinucleation,
nuclear hyperchromatism, eccentric position of the nucleus,
and cytoplasmic basophilia (5,6).
In response to these dilemmas, a range of special stains
have been empirically introduced to highlight various features of the morphology. The methods in diagnostic cytology
illustrate this point. At present, diagnostic cytology is typically based on a set of staining methods (7). This includes
the Papanicolaou stain (8,9) and the May-Grunwald-Giemsa
stain (or Romanowsky) (10); hematoxylin and eosin (H&E),
Shorr’s endocrine stain, and the Pappenheim method are less
frequently used (11). The role of these stains is to assist in
defining useful criteria that generally enable differentiating
neoplasia from non-neoplastic states.
More recent work focuses on histochemical, immunocytochemical, and/or molecular biological methods for detecting molecular markers (e.g., in situ hybridization). These
97
approaches are predicated on highlighting the unique functional biology believed to distinguish neoplasia from nonneoplastic states, e.g., differentiation and proliferation. Such
approaches also are useful for revealing the unique molecular
properties of specific cancer types, allowing their proper
identification or subclassification. Target molecules for these
techniques include lipids, carbohydrates, proteins, and nucleic
acids (e.g., RNA) (1,3,12–14).Two major limitations of such
strategies are well recognized. On the one hand, these molecular markers are informative for a particular tumor or class of
tumors, e.g., GFAP for astrocytic neoplasms (15), but are not
applicable to most carcinomas and sarcomas. On the other
hand, the same molecule is often expressed in reactive or normal cells of the same origin as the tumor, where the difference
is more quantitative than qualitative. This fact can make the
differential diagnosis of a florid reactive process versus cancer challenging (16). Although these adjunctive techniques
currently play an important role in routine detection and/or
classification of many forms of neoplasia, no single marker or
methodology has been described that is broadly applicable to
all, or even most neoplastic conditions. Hence, the quest is
ongoing for a universal cancer cell stain.
A central concept underlying efforts to develop a panmalignancy stain is that some features of oncobiology are
common to almost all cancers, namely, the altered states of
proliferation (17), metabolism (18–20), and differentiation
(26). Ideally, such a histochemical stain should testify to the
presence of a neoplastic phenotype by producing a distinct
heterochromatic or tinctorial outcome. For that reason, the
introduction of a new histochemical stain, the CellDetect®
technology, that claims to tinctorially differentiate cancer from
non-neoplastic cells, may represent a significant advance.
We present a critical investigation of this novel staining
procedure as applied to cultured cell lines as well as clinical
material. The goal was to show that the Zetiq CellDetect®
stain imparts a distinct red/purple color to the cytoplasm of
neoplastic that clearly distinguishes them from green/blue
stained non-neoplastic cells and tissues. The findings can be
objectively quantified, and reproducibly demonstrated in
cell cultures with an enzyme-linked immunoassay (ELISA)
reader detecting the two appropriate wavelengths of the red/
neoplastic and green/non-neoplastic tinctorial phenotypes.
A preliminary nonblinded series of biopsies and cytological
preparations also were examined to compare the consistency
of detecting neoplastic cells with CellDetect® to the cytologically identified neoplasia based on traditional H&E- or
Pap-stained preparations. In regard to such preparations, the
stain also affords the ability for morphological assessment
similar to standard diagnostic stains. The results of these
investigations suggest that the CellDetect® staining technology exhibits a unique capability for detecting neoplasia, and
appears practical for routine diagnostic and experimental
applications.
Materials and Methods
Study Material
Cell Lines
Source. HT-29 human colon carcinoma cells and HeLa
human cervical carcinoma were purchased from American
Type Culture Collection (ATCC).
Culture protocol. HT-29 cells (ATCC number HTB-38)
were grown in McCoy 5A medium with 1.5 mM L-glutamine
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supplemented with 10% fetal bovine serum, 100 units/mL
penicillin, 0.1 mg/mL streptomycin, 12.5 units/mL Nystatin,
and 2200 mg/L sodium bicarbonate. The cells were seeded
in 96-well plates at density of 80,000 cells/well, 100 µL per
well. HeLa cells were grown in MEM-Eagle medium with 2
mM L-glutamine and Earle’s BSS adjusted to contain 1.5
g/L sodium bicarbonate, 0.1 mM nonessential amino acids,
1.0 mM sodium pyruvate, 10% fetal bovine serum, 100
units/mL penicillin, 0.1 mg/mL streptomycin, and 12.5 units/
mL Nystatin. The cells were seeded in 96-well plates at density of 25,000 cells/well, 100 µL per well.
Forced Differentiation
After 24 h of incubation in medium, half of the wells were
treated with 10 mM of sodium butyrate (Sigma B-5887) and
incubated for 48 h. This forces the cells to a differentiated
phenotype (27,28). The medium was replaced with serumfree medium with and without 10 mM of sodium butyrate,
and the plates were incubated for an additional 24 h. The
total time of sodium butyrate treatment was 72 h. The cells
were then washed once by serum-free medium without
sodium butyrate, incubated for half an hour, dried at room
temperature for 24 h, and stained.
Biopsies
Serial sections were cut 4 microns thick from formalinfixed, paraffin-embedded biopsies of uterine cervix and urinary bladder. Each set of sections was divided into two
staining groups; one group was stained with hematoxylin
and eosin (H&E), and the other group was stained with
CellDetect® (Zetiq Technologies, LTD, Ramat Gan, Israel).
The material included samples with foci of carcinoma
(cervix = 10, bladder = 5, total = 15), and squamous dysplasia/in situ neoplasia (n = 15). Non-neoplastic, normal or
metaplastic foci of squamous epithelium were present in all
cervical biopsies. Microscopic examination of H&E-stained
slides from each case confirmed each diagnosis.
Cytological Preparations
Cervical smears prepared by ThinPrep® technology
(Hologics, Bedford, MA) were stained with the Papaniculou
stain (LEM Pathology Laboratory, Nes-Ziona, Israel) and
CellDetect® stain.
Staining Protocols
H&E Staining Protocol
Paraffin-embedded, 4-microns-thick tissues on slides were
deparaffinized in two xylene baths, 3 min each, and washed
in two consecutive ethanol baths, 3 min each, followed by
two consecutive 95% ethanol baths, 3 min each, and a distilled water bath, for 3 min. After deparaffinization, the
slides were dipped in hematoxylin stain (Dye-1) for 1 min
and washed in tap water (running in the container) until the
water was clear. Then, slides were immersed in eosin stain
(Dye-C) for 1–2 min, and washed in tap water as described
previously. The slides were then dehydrated in ascending
alcohol solutions (50%, 70%, 80%, 95% × 2, 100% × 2) and
cleared with xylene (3–4 times). The dehydrated slides were
mounted with Permount or another suitable organic mounting medium.
Papanicolaou Staining Protocol
Smears were dried, and the following staining and washing steps were taken: Slides were washed with 70% ethanol
Colorometric Detection of Cancer / Idelevich et al
for 2 min and then twice with tap water for 1 min each. Slides
were stained with Gill’s hematoxylin for 4 min and rinsed
with Scott’s water for 2 min, washed with 70% ethanol for 1
min, and then with 95% ethanol for 1 min. Then slides were
stained with the use of Orange G for 2 min and washed twice
with 95% ethanol for 1 min each. Then the slides were
stained for 10 min with Eosin A (Eosin Azure), which is a
counterstain, comprising the dyes Eosin Y, Light Green SF
yellowish, and Bismarck brown Y. Slides were then washed
twice with 95% ethanol for 1 min each, and treated twice
with xylene for 5 min each. Slides were mounted and, once
dry, screened under the microscope.
Zetiq CellDetect® Staining
Three types of preparations were used, and each required
a specific kit optimized for that particular type of preparation. The three kits used were CellDetect® biopsy staining
kit; CellDetect® cervical cytology staining kit; and the
CellDetect® tissue culture staining kit. The staining was performed according to the manufacturer’s instructions.
Although the details of the protocol and the formulation of
the stains are currently proprietary information, the kits contain three ingredients: 1) an acidophilic red stain, 2) a basophilic green stain, and 3) a proprietary plant extract necessary
for the specific reaction. The protocol involves 6 steps,
which can be completed in approximately 30 min.
Reproducibility of CellDetect® Staining
Two experiments were performed; one involved cell culture and the other biopsies. In the first experiment, reproducibility of tinctorial differences between CellDetect®-stained
cultures (wells) with malignant or differentiated phenotypes
was assessed; 30 cultures per treatment group were used.
After staining with the CellDetect® protocol, absorbance was
read using an ELISA reader (Genius by Tecan, San Jose, CA)
at 540 nm and 630 nm for each well. An ELISA reader is a
wavelength-specific light detection system used for quantifying assays based on the color of the product. The ratio between
the absorbance of the red dye (540 nm) and the green dye
(630 nm) was calculated. A ratio larger than 1.0 reflected red
staining, and a ratio less than 1.0 reflected green staining.
In the second experiment, serial sections from three cervical biopsy blocks (one normal, and two CIN3 cases) were
stained on different days (2–3 d apart for each test) and by
independent operators, and photographed using a light
microscope. The slides were assessed for equivalence of
stain quality and hue. Representative actual microphotographs are illustrated in Results.
Statistical Analysis
Means and SD were computed for ELISA reading results
of experiments investigating the tinctorial difference between
malignant phenotype and differentiated phenotype in tissue
culture. The statistical significance of the differences in the
mean were analyzed using the Student t test.
Results
Cultured Cancer Cell Lines
Staining Reaction for Cells with Malignant Phenotype
The colon carcinoma cell line (HT-29) and HeLa cell line
(cervical cancer) exhibited a diffuse, uniform red/purple tint
to their cytoplasm. This is illustrated for the HT-29 cells
(Figure 1A and Figure 2, top). Using an ELISA reader, these
color changes were objectively documented.
Staining Reaction after Forced Differentiation
The effect of sodium butyrate on many transformed cell
lines is to force them to differentiate. This experiment was
performed on the HeLa cells and HT-29 cells, which produced a consistent and dramatic shift of staining. Cytoplasm
of cells after differentiation stained green/blue in both types
of cultures.
Histological Preparations
Comparison of Squamous Cell Carcinoma (SCC) Biopsies
with Non-Neoplastic Squamous Epithelium
For this part of the study, 15 cases of cervical SCC based
on standard pathological criteria in H&E preparations
(Table 1) were compared with 15 non-neoplastic regions of
Figure 1. Effect of forced differentiation on CellDetect® staining in HT29 cells. The cells without butyrate treatment have a malignant phenotype and stain
red (A). Butyrate treatment (B) induced differentiation of HT29 cells and changed cytoplasmic staining to green, similar to other normal (Figure 5) and reactive cell populations (Figure 6). Magnification ´167.
The Journal of Histotechnology / Vol. 32, No. 3 / September 2009
99
normal or metaplastic epithelium, they also appeared blue.
This finding emphasizes that the cytoplasm is the diagnostically-sensitive cell domain in these preparations.
Comparison of SCC Biopsies with Dysplasia
Cervical dysplasia/in situ neoplasia confirmed by H&E
were CellDetect® stained. Here, too, the histochemical process produced red-purple cytoplasm (Figure 7, CIN2), as in
cases of carcinoma, described previously.
Figure 2. Graph demonstrating reproducibility of staining between
butyrate-differentiated and malignant HT29 cells. After CellDetect® staining, absorbance was read spectrophotometrically at 540 nm and 630 nm for
each well (n = 30). The ratio between the absorbance of the red dye (540 nm)
and the green dye (630 nm) was calculated. A ratio larger than 1.0 reflects
red staining, and a ratio less than 1.0 reflects green staining. The two sets of
experimental results are clearly consistent and separated in all instances.
squamous epithelium. The features assessed for samples
from both groups are listed in Table 1. The table highlights
that the CellDetect® stain not only discriminates neoplasia
from non-neoplasia based on tinctorial differences, but also
enables characterizing the purely morphological aspects of
the neoplastic phenotype.
Our observations show that all cases that satisfy the morphological criteria for SCC have clear red/purple cytoplasm
staining with CellDetect® (Figure 3). Staining of transitional
cell carcinoma of the urinary bladder (Figure 4) was comparable to the examples of squamous neoplasia. The neoplastic
epithelial cells clearly demonstrated the red/purple cytoplasmic reaction. Note also the parallelism in detecting the characteristic morphological criteria in both CellDetect® and
H&E.
Sections from cervical epithelium that was non-neoplastic
when examined with H&E were stained by the CellDetect®
method. As seen in Figure 5, CellDetect®-stained preparations of normal/non-neoplastic cells exhibited cytoplasm
that was blue/green stained, similar to the findings for differentiated cell lines (Figure 1B). The morphologic features of
CellDetect®-stained non-neoplastic cervix cells was comparable to that seen in the H&E stain (Table 1). Metaplastic
foci (Figure 6) also featured cells with blue/green cytoplasm,
as in the normal epithelium (Figure 5).
It is worth emphasizing that the color of the nuclei was not
diagnostic; nuclei were typically red, but in some cases of
Cytological Preparations
All preparations included normal and abnormal cells. For
Pap-stained cells, a range of cytoplasmic color values were
evident that lacked any consistent relation to neoplastic status as reflected in the cytomorphology (Figure 8B, D). In
contrast, CellDetect®-stained cells exhibited a consistent
relation to cytomorphology. Morphologically “normal” cells
have a greenish cytoplasm, whereas morphologically “abnormal” cells exhibit reddish cytoplasmic staining (Figure 8A,
C) as seen in neoplastic tissues (Figures 3, 4, 7, and 9) and
cell cultures (Figure 1).
Reproducibility
Another basic issue raised by these observations is the
extent of reproducibility. In a preliminary way, this question
was assessed in two experiments, one involving cell cultures
and the other biopsies.
Cell Cultures
Figure 2 shows a graphic summary of staining results with
CellDetect® for malignant phenotype versus differentiated
phenotype. The results for all 30 cultures in each treatment
group were tinctorially monomodal. The differentiated cultures have a ratio of OD 540/630 in the green range (mean,
SD: 0.79 ± 0.04), whereas the malignant phenotype is consistently restricted to ratio in the red range (mean, SD: 2.23 ±
0.14). The differences in the two groups are highly significant (t-test, P < 0.0001). The graphs make two additional
points: first, that one can instrumentally quantify the tinctorial CellDetect® reaction in cultured cells and not merely rely
on subjective microscopic observations; second, the staining
reaction, both in phenotypically differentiated and malignant
cells, is highly reproducible. Hence, the reaction in this system is consistent and reproducible. The same experiment was
replicated in different plates, on different days, by different
operators on over 10 occasions with identical outcomes.
Biopsies
Two aspects of reproducibility of staining in tissue sections were addressed. First, staining was replicated on three
Table 1. Comparison of CellDetect® and H&E staining in relation to malignant and non-neoplastic tissues
and their morphological features
Nuclear/cytoplasm ratio
Prominent nucleoli
Irregular nuclear contours
Nuclear color
Cytoplasm color
Intensity of cytoplasmic stain
a
b
Non-neoplastic
H&Ea
Low
Absent
Absent
Blue
Pink
Uniform
Non-neoplastic
CellDetect® stain
Low
Absent
Absent
Blue/sometimes red
Blue/green
Uniform
Neoplasia H&E
High
Present
Present
Blue
Pink
Uniform
Neoplasia CellDetect®
stainb
High
Present
Present
Red
Red/purple
Uniform
Non-neoplastic, n = 15.
Neoplasia cases, n = 15.
100
Colorometric Detection of Cancer / Idelevich et al
Figure 3. CellDetect® (A) and H&E (B) staining of squamous cell carcinoma of cervix. The malignant cells in CellDetect® preparations have uniform red/
purple cytoplasm, which is also present in in situ neoplasia (Figure 7). Magnification ´333.
sequential sections from the same block, on different days
by the same operator. The results are illustrated in Figure 9,
for a case of cervical intraepithelial neoplasia. The three
photomicrographs are nearly identical, showing the same
linear tinctorial pattern of neoplastic and non-neoplastic cellular zones. Each zone stained comparably in terms of the
intensity and hue of the colors. Second, the reproducibility
was assessed for different operators; the outcomes were also
the same (results not shown). Collectively, these observations indicated that the staining technology produced highly
reproducible results for tissues and cells.
Discussion
In this study, a novel histochemical stain for detecting
neoplasia, developed by Zetiq, was assessed for its performance at the light microscopic level. The principal question
in this initial trial was whether the Zetiq CellDetect® stain
produced a tinctorially specific and reproducible reaction in
neoplastic cells versus non-neoplastic cells. By using the cell
lines, we also had the opportunity to objectively quantitate
the staining performance in a single model system with a
biphasic phenotype that can be experimentally switched.
Two different epithelially derived cell lines were used, so the
outcomes are not likely to be tissue- or cell-type specific. By
the use of a wavelength-specific light detection system
(ELISA reader) the clear-cut distinction between neoplastic
and non-neoplastic phenotype was readily and objectively
discriminated. This was true over numerous replications. The
staining reaction was specific to the cytoplasm; neoplastic
cells had red/purple stained cytoplasm in contrast to nonneoplastic cells with blue/green stained cytoplasm. In preliminary work, this was found in several additional cell lines,
suggesting applicability to a broader range of cell types.
To insure that the staining phenomenon was not limited to
in vitro conditions, a preliminary nonblinded or stratified
evaluation of clinical material was undertaken. The results
in these biopsies and cytology preparations were similar to
the more systematic and dynamic in vitro findings. Samples
diagnosed as neoplasia—of different degrees and tissueThe Journal of Histotechnology / Vol. 32, No. 3 / September 2009
types based on H&E or Pap-stained criteria—always had a
red/purple stained cytoplasm.
In a practical vein, we noted that staining for different
types of preparations was highly reproducible, and required
only 30 min to complete. Additionally, the staining outcomes
Figure 4. Example of CellDetect® staining of transitional cell carcinoma of
the urinary bladder. Neoplastic cells have distinct red/purple cytoplasm.
Magnification ´ 333.
101
Figure 5. CellDetect® (A) and H&E (B) staining of normal cervix. Normal cervical epithelia cells after CellDetect® staining exhibit blue/green cytoplasm,
which is the diagnostic cellular compartment. Nuclei of epithelial cells typically stain red, but may occasional be blue-green. Magnification ´333.
were consistent colorometrically, across samples, so that the
presence of a malignant phenotype could be correctly determined by wavelength-specific absorbance assessment of the
preparations, even independent of an observer. It is likely that
any instrument that analyzes the optical properties of transmitted light could be similarly used. For these reasons, the
method should be readily adaptable to high-throughput screening and automated analyses, as well as point of care testing in
settings where traditional triage mechanisms are not available.
However, the ability to colorometrically track the phenotypic
dynamics of cancer cell lines in vitro would also make this
approach directly applicable to experimental oncology.
The most provocative observation in the current work was
the consistent ability of the CellDetect® stain to differentiate
cancer from non-neoplastic states, as expressed phenotypically in cell culture or by standard criteria in H&E- or Papstained preparations. Although this initial series of samples
consisted of epithelial lineage phenotypes, preliminary work
on other cell types, e.g., glial, have produced identical results
(data not shown). From this collective experience, we tentatively suggest that the CellDetect® staining technology will
exhibit a uniquely broad scope in detecting neoplasia independent of cell type of origin. In addition, as highlighted previously, the CellDetect® technology provides tinctorial as well
as morphological information. With this new stain, nuclear
criteria for neoplasia were easily observed, as were other key
cytologic features, providing a joint morphological and histochemical profile on putative cancer cells. This interesting
Figure 6. CellDetect® (A) and H&E (B) staining of non-neoplastic mature metaplasia. Cytoplasm of metaplastic cells stained blue/greenish, similar to normal cells (Figure 5). Magnification ´333.
102
Colorometric Detection of Cancer / Idelevich et al
Figure 7. CellDetect® (A) and H&E (B) staining of cervical intraepithelial neoplasia (CIN2). In contrast to the findings on normal cells in Figure 5, neoplastic
cells have reddish/purple cytoplasm in CellDetect® stained sections. H&E cytoplasmic staining fails to tinctorially distinguish non-neoplastic cells (Figure 6)
from CIN cells. Magnification ´333.
suggestion justifies a few comments regarding the possible
biological substrate(s) for such an effect. Given the bipolar
nature of the histochemical cytoplasmic reaction, we hypothesize that the findings reflect fundamental properties that distinguish the neoplastic state, in general. In such global terms,
neoplasia typically exhibits striking alternations in the state of
metabolism, level of differentiation and rates of proliferation.
In terms of our own observations, an important set of
directly relevant findings derive from the forced differentiation experiments in tissue-cultured cell lines. The
change in color to a “normal” staining phenotype that characterizes the differentiated state of treated cultures stands in
sharp contrast to the “malignant” type staining reaction in the
naïve cultures. These results would support the hypothesis
Figure 8. CellDetect® (A, C) and Pap (B, D) staining of cervical smears. These smear preparations contain cells with high-grade squamous intraepithelial
lesions (A, B) as well as low-grade squamous intraepithelial lesions (C, D). Color of cytoplasm in cells in Pap preparation is variable and does not
correlate with cytomorphology. In CellDetect® stained samples, the cytoplasm of normal cells stains green, whereas atypical cells have reddish cytoplasm.
Magnification ´333.
The Journal of Histotechnology / Vol. 32, No. 3 / September 2009
103
Figure 9. Reproducibility of CellDetect® staining technology in biopsies. Three adjacent sections from a biopsy of cervical intraepithelial neoplasia (CIN1)
were stained on 3 different days (A–C). The laminar patterning of tinctorial differences between neoplastic (red) and non-neoplastic (blue) cells is essentially
identical in regards to color intensity and hue. Magnification ´3 (A and B); ´4 (C).
that staining indicates the level of proliferation and/or differentiation of the cell population. Similarly, this was seen
in more differentiated fields within invasive squamous cell
carcinoma (data not shown). Nonetheless, caution is justified
before embracing this conclusion because of proliferative typically normal staining phenotype of proliferative basal cells
and metaplastic cells. In true neoplasia, CellDetect® apparently
stains an oncogenic factor absent from the latter. Clearly,
this issue deserves to be re-evaluated with the use of specific
markers indicative of proliferation and differentiation status.
Another prominent point of functional distinction between
neoplasia and normal tissues is the augmented metabolic
level in cancer cells. This is known as the “Warburg effect”
(18–20), which refers to the high rates of anaerobic glycolysis in cancer cells. The high rate of glycolysis causes a cascade of by-products that changes the metabolic status of the
cell, including its intracellular pH. It is known that cancer
cells display a more alkaline intracellular pH (26) due to
the differences in their metabolic status. Although this difference in intracellular pH is minor, it may be sufficient to
produce a significant histochemical change. Hence, the possibility that the CellDetect® stain detects a differential in
metabolic status is an interesting, testable hypothesis that
deserves further consideration.
In conclusion, the Zetiq CellDetect® staining technology
allows one to distinguish common forms of epithelial neoplasia from non-neoplastic homotypic tissues and cells, in a
rapid, consistent, reproducible manner. On the basis of the
current study and preliminary observations, we anticipate
that this histochemical approach will be applicable to a wide
range of diagnostic and experimental settings requiring
detection of neoplasia. We tentatively suggest that the stain
detects a generic cancer phenotype that could reflect metabolic, proliferation and/or differentiation states.
Acknowledgments
Pavel Idelevich, Ilia Rivkin, Dov Terkieltaub, and Adi
Elkeles are employees of Zetiq Technologies Ltd, through
104
which the CellDetect® products are marketed. Funds for this
work were provided by Zetiq Technologies Ltd.
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