a comparison of acridine orange and feulgen cytochemistry...

[CANCER RESEARCH 38. 1893-1898, July 1978]0008-5472/78/0038-0000$02.00

A Comparison of Acridine Orange and Feulgen Cytochemistry of HumanTumor Cell Nuclei1

James E. Gill, Leon L. Wheeless, Jr., Carol Manna-Madden,- Richard J. Marisa, and Paul K. Horan

Analytical Cytology Division, Department of Pathology, University of Rochester School of Medicine and Denistry, Rochester, New York 14642

ABSTRACT

Specimens of cells derived from tumors of the humanfemale genital tract plus normal cells as standards havebeen divided into aliquots and stained according to acri-dine orange or pararosanilin:Feulgen procedures. Acridine orange-stained cells were slit-scanned for 535 nmnuclear fluorescence; Feulgen-stained cells were comb-scanned for 580 nm nuclear absorbance. For each specimen examined, the tumor celhnormal cell ratio of meannuclear fluorescence following acridine orange stainingwas greater than the tumor celhnormal cell ratio of meannuclear absorbance following Feulgen staining. The tumorcelhnormal cell ratio of mean nuclear fluorescenceranged from 2.3 for a nonkeratinizing squamous cellcarcinoma to 3.9 for a keratinizing squamous cell carcinoma. The tumor celhnormal cell ratio of mean nuclearabsorbance ranged from 1.4 for a mixed mesoderma!sarcoma to 2.3 for a small cell squamous cell carcinoma.

These results indicate that the elevated nuclear fluorescence intensity from acridine orange-stained tumor cellscannot be explained solely on the basis of elevatedFeulgen:DNA content. An alternative hypothesis, consistent with these results, is that DNA is the principal bindingsubstrate for intranuclear acridine orange and that theDNA of certain tumor cells is more accessible to acridineorange than is the DNA of normal cells.

INTRODUCTION

Cells derived from many human malignant tissues havealtered chromatin content and structure. The cellular Feul-gen:DNA content of malignant tissues is generally elevated(1), and the chromatin patterns as seen under the lightmicroscope are different from those associated with normaltissues (9). Both of these generalizations hold true forcancers of the uterine cervix in particular (13).

Research efforts directed toward cytology automationhave attempted to use elevated DNA content (14) andaltered chromatin patterns (12) as markers for abnormalcells. Fluorescent dyes believed to bind to DNA within cellshave been tested for their ability to discriminate betweennormal and abnormal cells. In particular, AO3 has been

shown to provide effective discrimination between normaland abnormal cells when 535 nm nuclear fluorescence ofAO-stained cells has been measured by a slit-scan technique (19).

1These studies were performed under National Cancer Institute ContractN01-CB-53930.

2 Present address: Doctors' Laboratory, Cuyahoga Falls, Ohio.3The abbreviation used is: AO, acridine orange.Received October 6. 1977; accepted March 22, 1978.

The purpose of the research was to determine the cyto-chemical basis for this discrimination. Although it has beenreported that intracellular AO:DNA complexes fluorescegreen, while AO:RNA complexes fluoresce red, the actualcytochemical behavior of AO is complex (11, 21). Thestaining protocol used for the experiments reported herewas derived (20) from the protocol of Von Bertalanffy (17).It differs significantly from those (15) for which DNA andRNA specificity has been claimed. However, since AO bindsto DNA and since the DNA content of abnormal cells iselevated, it appeared plausible that the elevated nuclearfluorescence from AO-stained abnormal cells might reflectelevated DNA content (6). To test this hypothesis, we haveexamined the AO staining of DNase I- and RNase-treatedcells and have compared Feulgen:DNA values and AOnuclear fluorescence values of aliquots of the same clinicalspecimens. The results of these experiments indicate thatDNA is the primary substrate for AO binding within cellnuclei but contradict the hypothesis that the integrated 535nm (green) fluorescence of AO-stained cells provides ameasure of their relative Feulgen:DNA content.

MATERIALS AND METHODS

Collection and Preparation of Specimens. Tumor specimens were collected from surgery, surgical pathology, andautopsy services. Specimens and their sources are summarized in Table 1. Cells derived from solid tumors wererinsed from these tissues into clear Mucosol (Lerner Laboratories, Stamford, Conn.), syringed to break up clumps ofcells, and mixed with normal material if none was presentin the specimen. Cells taken by cervicovaginal scrape weresuspended immediately in clear Mucosol and combinedwith a suspension of normal cervical material. Normalspecimens were collected from an outpatient clinic.

Nuclease Treatment. Cells suspended in Mucosol werecentrifuged and resuspended in 20 mM citrate:phosphate,pH 3.0, with 0.1% Triton X-100 for 1 min to facilitatenuclease penetration (5). Cells were then rinsed in pH 7.5buffer, resuspended in buffer with appropriate salts, andbrought to 4 x 103 units/ml with DNase I (Sigma ChemicalCompany, St. Louis, Mo.) or 103 units/ml with RNase(Sigma). Cell suspensions were held at 37Â°Cfor 30 min. The

cells were then centrifuged and stained.AO Staining. An aliquot of the cell suspension was

centrifuged, and the pellet was resuspended in 2.5 x 10~5

M AO:0.2 M phosphate buffer, pH 7.O. After 10 min, the cellswere centrifuged, resuspended in 3.8%glutaraldehyde:0.14M phosphate buffer at pH 7, and stored in this suspensionuntil needed (3).

Pararosanilin:Feulgen Staining. A second aliquot of the

JULY 1978 1893

on July 3, 2019. © 1978 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

J. E. Gill et al.

Table 1Description of specimens analyzed

TumormaterialDiagnosis"Mixed

mesodermalsarcomaOvarian

Adeno-carcinomaSmall

cellsquamouscellcarcinomaKeratinizingsquamous

cellcarcinoma.metastatic

toliverNonkeratinizingsquamous

cellcarcinomaofcervixSarcoma;metastatic

fromamixedmesodermalsarcomaTissueSolid

andfluidSolidCervicalscrapeSolidSolidVaginalscrapeSourceSurgicalpathologyAutopsyOperating

roomAutopsySurgical

pathologyOperating

roomSource

of normal materialbClinicClinicClinicft

" For diagnostic, see Ref. 13.A No additional normal cells required.

cell suspension was centrifugea, 1 drop of the concentratewas placed on 1 slide, and "pull-apart" smears were gen

erated with a second slide. These slides were immersed inmethanol:formalin:glacial acetic acid (85:10:5, v/v) for atleast 1 hr and then taken through a Feulgen stainingprotocol (2).

Schiffs reagent was prepared according to the methodof Graumann (10) with the following modifications. Basicfuchsin (C.I. 42,500), 0.1 g, was dissolved in 15 ml of 1 NHCI and then combined with 85 ml of 2.6 x 10~2 M potas

sium metabisulfite in distilled water and stored overnight inthe dark in a capped Teflon bottle. The solution was shakenwith activated charcoal (Darco G-60) for 2 min and then

filtered through a Millipore prefilter. The resulting stainingsolution was clear and nonfluorescent.

Analyses of Stains. Absorption spectra of stains wereobtained with a Beckman Acta V spectrophotometer andcompared with published spectra (22) to verify identityand purity. Fluorescence analysis of stains were madewith an Aminco-Bowman spectrophotofluorometer modified for photon counting (8). Thin-layer chromatographyof "purified" AO (Polyscience, Inc., Warrington, Pa.)

on silica or on cellulose with a solvent mixture ofbutanol:ethanol:H,O:NH4OH (32:10:10:0.3, v/v) revealed 1minor impurity. Other commercial samples of AO had substantial contamination. The "purified" AO was used without

further purification.Slit-Scan Measurements of Integrated Nuclear Fluores

cence. Slit-scans of individual AO-stained cells were recorded with a Zeiss Axiomat microscope-photometer system, equipped for both epi- and transmitted illumination.For epi-illumination, the light source was an Osram XBO-

150 xenon arc lamp. Exciter filters were KP 490 and KP 500

in series; the reflector transition wavelength was 510 nm,and the barrier filters were an LP 528 and a 525 to 566 nmband pass (full width at half-maximum); green fluorescencewas selected for measurement. The objective was x50 witha numerical aperture of 0.95; effective slit width was 5 /nm.Epi-illumination intensity was reduced to approximately 5%

for location and positioning of cells (to avoid bleaching thespecimen) and then increased to the maximum for slitscanning. Scanning of the stage and collection of datawere controlled by a POP 11/40 computer (Digital Equipment Corp., Maynard, Mass.). The fluorescence intensityprofile was collected by the computer and analyzed for celldiameter, nucleus diameter, ratio of nucleus diameter tocell diameter, integrated nuclear fluorescence, and integrated cell fluorescence (18).

Comb-Scan Measurements of Integrated Nuclear Ab-sorbance. Absorbance measurements of Feulgen-stained

nuclei were obtained with the Axiomat system. For transmitted illumination, the light source was a tungsten:halogenlamp operated at 12 Vd.c. A linear wedge interference filterwas used to select a band of light centered at 580 nm. Thefield stop diameter was 0.16 mm, giving a 34-/j,m-diameter

circle of illumination in the specimen plane. The objectivewas x50 with a numerical aperture of 0.95 to provide aneffective photometer spot aperture with a diameter of 0.5tÂ¿min the specimen plane. The image was comb-scanned

across this spot aperture by stepping the scanning stage in0.5-/Â¿mincrements at approximately 150 Hz under control

of the computer. Light passing through the aperture wasdetected by a photomultiplier tube, and the photomultiplieroutput was converted to a voltage. This voltage was recorded by the computer after each increment of the stage.The average value from the first line of the comb scan wascalculated and considered as background intensity, /â€ž.Foreach succeeding increment, the computer recorded themeasured intensity, /,/, and calculated the specimen ab-sorbance of that position as A' = log (V/.Â«1')- The total

absorbance of the scanned nucleus was calculated as thesum of all A'. Duplicate comb-scan measurements were

made of specimen nuclear absorbance, averaged if thevalues agreed within 20%; otherwise, they were discarded,which was rare. Single slit-scan measurements were made

of nuclear fluorescence, since each measurement causedpartial fading of fluorescence.

Before specimens were measured for either nuclear absorbance or fluorescence, the absorbance of a marked"standard" Feulgen-stained nucleus was measured repeat

edly, and the mean and standard deviation was checkedagainst previous measurements of this nucleus to verifyperformance of the system. (Standard deviations were typically 5 to 6% of the mean.)

Statistical Analysis and Protocol. Analysis of the datawas based on the following assumptions: (a) nuclear fluorescence from slit-scan measurements and nuclear absorbance from comb-scan measurements constitute random

variables; (b) each of these random variables is associatedwith a probability density function such that mean andstandard deviations exist; (c) if A is the mean of theintegrated absorbance of abnormal cell nuclei and 8 is themean of the integrated absorbance of normal cell nuclei,

1894 CANCER RESEARCH VOL. 38



Cytochemistry of Human Tumor Cell Nuclei

Obtain Tumor Tissue

Form Suspension of Single Cells

Seed with Normal Cells if Required

Divide Suspension into Two Aliquots

Aliquot 1

i

Apply Cells to Slides

I

Fix in MFG

I

Feulgen Stain

I

Comb Scan for NuclearAbsorbance

IComputer Analysis andHistogram of Feulgen-

DNA Distribution

Aliquot 2

I

Stain with AO

I

Fix in Glutaraldehyde

i

Apply Cells to Slides

ISlit-Scan for NuclearFluorescence

iComputer Analysis andHistogram of NuclearFluorescence Distribution

Chart 1. A flow diagram of the basic experimental protocol. See "Materials and Methods" for details. MFG, methanol:formalin:glacial acetic acid

(85:10:5, v/v).

then the ratio of the means, R = Ã„/B, is also a randomvariable; (d) repeated measurements of R would be normally distributed; (e) if nuclear fluorescence from AO-stained cells represents Feulgen:DNA content, then theratio of the means of abnormal and normal nuclear fluorescence will be the same, statistically, as the ratio of themeans of abnormal and normal nuclear absorbance; (f) theappropriate test for significant difference in the ratios of themeans is the f test (23).

The basic protocol for the experiments with human material is given in Chart 1.

Isolation and Staining of Rat Liver Nuclei. Isolationfollowed the procedure of Umana and Dounce (16) with thefollowing modifications. Solutions of 0.44 M sucrose contained 1 mw MgCI2; the centrifugation through the 2.2 Msucrose was carried out in a Beckman J21C centrifuge withan SW-13 rotor spun at 13,000 rpm for 1 hr. The finalnuclear pellets were resuspended in 10 ml of 0.44 M sucrose, and 10 ml of 20% formalin in 0.44 M sucrose wereadded and mixed by vortexing. The nuclei were fixed in thismedium overnight and then stained according to a pararo-sanilin:Feulgen procedure for cell suspensions (7).

Flow Cytometry of Rat Liver Nuclei. Suspensions ofFeulgen-stained nuclei in distilled water were diluted withdistilled water to a concentration of about 106 nuclei/ml.The analysis and sorting were carried out with a CoulterModel TPS-1 sorter (Coulter Electronics, Hialeah, Fla.).

Table 2Mean nuclear fluorescence of normal and tumor cells after AO staining

SpecimenMixed

mesodermal sarcomaOvarian adenocarcinomaSmall cell squamouscellcarcinomaKeratinizing

squamouscellcarcinomaNonkeratinizing

squamouscellcarcinomaSarcoma,

metastaticNormal"100

100100100100100Â±24"

Â±34Â±24Â±

26Â±

12Â±

14(22)c

(30)(27)(21)(24)(30)272

330274386229346TumorÂ±

90Â±161

Â±115Â±

219Â±

98Â±

220(149)

(52)(54)(55)(32)(33)2

32323Ratio

ofmeans.72

.30

.74.86.29.461Â±Â±Â±111212.11

.95

.32.42.02.26

" The mean of the normal cell nuclear fluorescence is normalized to 100 for eachspecimen. The mean of the tumor cell nuclear fluorescence is normalized by the samefactor.

b Mean Â±S.D.c Numbers in parentheses, number of cells measured.

Table 3Mean nuclear absorbance of normal and tumor cells after pararosanilin:Feulgen staining

SpecimenMixed

mesodermal sarcomaOvarian adenocarcinomaSmall cell squamous cell

carcinomaKeratinizing squamous cell

carcinomaNonkeratinizing squamous

cell carcinomaSarcoma, metastaticNormal"100

Â±29.46 (28)Â°

100 Â±21.3 (45)100 Â±14.7(28)100

Â±19.2 (33)

100 Â±26.0 (25)

100 Â±22.0 (29)Tumor141

Â±59.2(25)172 Â±83.3(50)227 Â±59.8(63)225

Â±101 (31)

184 Â±62.4(30)

151 Â±76.4(29)Ratio

ofmeans1

.41 Â±0.721.73 Â±0.912.27 Â±0.682.25

Â±1.10

1 .84 Â±0.62

1 .52 Â±0.83

" The mean of the normal cell nuclear absorbance is normalized to 100 for each specimen. Themean of the tumor cell nuclear fluorescence is normalized by the same factor.

* Mean Â±S.D.c Numbers in parentheses, number of cells measured.

JULY 1978 1895



J. E. Gill et al.

Histograms of frequency versus integrated fluorescenceintensity were obtained (4). The light scatter channel wasused to gate out fluorescence signals from small bits offluorescent debris. Sorted nuclei were examined to verifythat peaks in the histogram corresponded to fluorescencefrom intact single nuclei.

Quantitative Microscopy of Rat Liver Nuclei. For measurements of nuclear fluorescence or absorbance with theAxiomat photometer system, 1 drop of the stained rat livernuclei suspension was smeared on a glass slide and allowed to air dry. The smear was covered with 1 drop ofCargille's type A immersion oil and coverslipped. Individual

nuclei were slit-scanned for integrated fluorescence orcomb-scanned for integrated absorbance as described

above. Statistical analysis of the ratio of tetraploid to diploidfluorescence or absorbance was as described above.

RESULTS AND DISCUSSION

Mean values for normal and tumor cell absorbance orfluorescence and the tumor celhnormal cell ratio of themeans have been calculated for each of the 6 specimenstested. Nuclear fluorescence data are summarized in Table2; nuclear absorbance data are summarized in Table 3. Ifnuclear fluorescence from AO-stained cells represented

nuclear Feulgen:DNA content, then the ratio of the meansof nuclear fluorescence would be indistinguishable fromthe ratio of the means of nuclear absorbance. Statisticalanalyses of the distributions, summarized in Table 4, indicate that in every case this "null" hypothesis, namely, that

the ratios are statistically indistinguishable, can be rejected

Ã¬u.

ls

30

25

20

15-

ID

S'

O 500

NUCLEAR FLUORESCENCE

20

15-

IO-

5

O 500

NUCLEAR ABSORBANCE

Chart 2. Nuclear AO fluorescence and nuclear Feulgen absorbance distributions for normal epithelial cells (open bar) and cells derived from a mixedmesodermal sarcoma (stippled bar). Both fluorescence and absorbance arein arbitrary units, normalized so that the mean value for normal cells is 100.Each bar is 40 units wide: both axes are linear.

with a confidence level of at least 95%.Histograms of data for 2 of the specimens tested are

shown in Charts 2 and 3. In each chart A represents thenuclear fluorescence distributions of normal and abnormalcells, and B represents the Feulgen absorbance distributions. Chart 2 represents the mixed mesodermal sarcoma(Tables 1 to 4, Line 1) while Chart 3 represents the smallcell squamous cell carcinoma (Tables 1 to 4, Line 3).

Nuclease treatments of normal clinical specimens wereperformed to test whether AO staining of nuclei was specificfor DMA. DNase treatment of cells reduced the nuclearfluorescence below the level of detection by the slit-scantechnique. From observation of slit-scan contours, residual

nuclear fluorescence of the DNase treatment is estimatedto be less than 10% of the value for untreated cells.Fluorescence microscopy of treated cells revealed a lack ofnuclear chromatin-like structures. Any remaining nuclear

fluorescence was associated with the nuclear membrane.RNase treatment of cells left the nuclear fluorescencestatistically indistinguishable from that of untreated cells.

These data indicate that AO staining of normal cell nucleiis 90% or more specific for DNA and does not involvenuclear RNA. However, these data do not indicate whetherthe amount of nuclear fluorescence from AO-stained cells

represents the amount of DNA present. The variability ofnuclear fluorescence from normal AO-stained cells (19)

indicates that factors other than DNA content must play arole. In short, specificity does not indicate stoichiometry.Experiments involving nuclease treatments of tumor cellsare pending.

It was necessary to test the possibility that the Axiomatsystem was providing fluorescence or absorbance meas-

ID

0JO 500

NUCLEAR FLUORESCENCE

30-

25-

20-

I5

IO

5

O 500NUCLEAR ABSORBANCE

Chart 3. Nuclear AO fluorescence and nuclear Feulgen absorbance distributions for normal polymorphonuclear leukocytes (open oar) and cellsderived from a small cell squamous cell carcinoma (stippled bar).




Cytochemistry of Human Tumor Cell Nuclei

Table 4Confidence levels for rejection of hypothesis that ratios of means

are indistinguishable

SpecimenMixed

mesodermalsarcomaOvarianadenocarcinomaSmall

cell squamouscellcarcinomaKeratinizing

squamouscellcarcinomaNonkeratinizing

squamouscellcarcinomaSarcoma,

metastaticf6.464.651.973.511.984.47Confidence

level0.99950.99950.950.9950.950.9995

Â§

1000

800

600

400

200

O,20 100 12040 60 80

CHANNEL NUMBER(RELATIVEFLUORESCENCEINTENSITY)

Chart 4. A flow cytometry histogram of frequency versus relative fluorescence for pararosanilin:Feulgen-stained rat liver nuclei. Relative fluorescence is the putative relative DMAcontent.

Table 5Mean absorbance and fluorescence of Feulgen-stained rat liver nuclei

Property measuredAbsorbance"

Fluorescence0Diploid100Â±12.5* (9)'

100 Â±18.0 (20)Tetraploid197Â±19.3(11)

214 Â±18.6(26)Ratio

ofmeans1

.97 Â±0.312.14 Â±0.43f1.28

" The mean of the diploid nuclei absorbance is normalized to 100, and the mean of thetetraploid nuclei is normalized by the same factor.

6 Mean Â±S.D.' Numbers in parentheses, number of nuclei measured.dThe mean of the diploid nuclei fluorescence is normalized to 100, and the mean of

the tetraploid nuclei is normalized by the same factor.

urements that were not proportional to the fluorescence orabsorbance of the stained nuclei. For example, the measurements might have indicated that 1 nucleus had 1.5 timesthe absorbance of a second nucleus when in fact the firstnucleus had twice the absorbance of the second. To makesuch a test, we analyzed Feulgen-stained rat liver nuclei forboth fluorescence and absorbance. Additionally, thestained rat liver nuclei were analyzed and sorted on thebasis of fluorescence intensity with a flow cytometer.

Pararosanilin:Feulgen staining of nuclei or cells generates both fluorescence and absorbance with the integratedintensities proportional to nuclear DMA content (2, 7). Ratlivers contain both diploid and tetraploid cells; hence,fluorescence or absorbance measurements of Feulgen-stained rat liver nuclei can be expected to generate bimodaldistributions, with the mean of the high-intensity peak equalto twice the mean of the low-intensity peak. Flow cytometermeasurements of fluorescence from the rat liver nucleigenerated the histogram shown in Chart 4. The ratio of themeans of the 2 peaks, high intensityilow intensity, is 2.06Â±0.45 (S.D.).

Measurements of absorbance or fluorescence from another aliquot of these rat liver nuclei, made with the Axiomatsystem, are summarized in Table 5. The ratios of the meansof absorbance and fluorescence data are not significantlydifferent from 2.0. This result shows that the Axiomatsystem does provide measurements that are proportional tothe fluorescence or absorbance of stained nuclei over theintensity ranges of interest. The differences between thefluorescence and absorbance ratios of means in Tables 2and 3 are not due to measurement artifact.

Thus, for each clinical specimen tested, the data indicatethat nuclear fluorescence from AO-stained cells is notproportional to Feulgen:DNA content but does provide ameasure of some other nuclear property associated withabnormality. If DNA is assumed to be the substrate for AObinding within the tumor as well as normal cell nuclei, itappears that this other property may be increased DNAaccessibility within the chromatin of tumor-derived cells.

In work to be published elsewhere,4 it is shown that

propidium iodide as well as AO provides discriminationbetween normal and tumor cells, based on measurementsof nuclear fluorescence. The data indicate that intercalativebinding of these fluorochromes occurs in both normal andtumor cells, providing further evidence that DNA is thebinding substrate for AO in normal and tumor cells.

An important practical result of these findings, relevantto cytology automation, is that integrated nuclear fluorescence at 535 nm from AO-stained tumor cells provides abetter indication of abnormality than does Feulgen absorbance (or any other measure of Feulgen:DNA content).

In summary, the increase in 535 nm nuclear fluorescencefrom AO-stained tumor cells is greater than the increase in580 nm absorbance of Feulgen-stained tumor cells, relativeto values for normal cells, with a confidence level of at least95%. This result can be accounted for on the basis ofincreased accessibility of DNA to AO within the chromatinof tumor cells.

4J. E. Gill, L. L. Wheeless, Jr., C. Hanna-Madden. and R. J. Marisa. Cyto-fluorometric and Cytochemical Comparisons of Normal and AbnormalHuman Cells, manuscript in preparation.

JULY 1978 1897



J. E. Gill et al.

ACKNOWLEDGMENTS

We thank Margaret Gambier and Marcia Zach for collecting the specimensused in these studies and Thomas Schmitt for supplying the rat liver.

REFERENCES

1. Bohm, N.. and Sandritter, W. DMAin Human Tumors: A CytophotometricStudy. Current Topics Pathol., 60. 151-214, 1975.

2 Bohm, N., and Springer, E. Fluorescence Cytophotometry: A ValuableMethod for the Quantitative Determination of Nuclear Feulgen-DNA.Histochemistry, 76: 100-118, 1968.

3. Cambier, M. A.. Wheeless, L. L., Jr., and Patten. S. F., Jr. A New Post-Staining Fixation Technique for Acridine Orange. Acta Cytol , 21: 477-480, 1977.

4. Crissman. H. A., Mullaney, P. F., and Steinkamp, J. A. Methods andApplications of Flow Systems for Analysis and Sorting of MammalianCells. Methods Cell Biol.,9: 180-242, 1975.

5. Darzynkiewicz, Z.. TrÃ¡ganos, F., Sharpless, T., and Melamed, M. R.Lymphocyte Stimulation: A Rapid Multiparameter Analysis. Proc. Nati.Acad. Sei. U. S., 73: 2881-2884, 1976.

6. Gill, J. E., Moran, P. K., and Wheeless. L. L., Jr. Cylofluorometric andCytochemical Comparisons of Normal and Abnormal Human Cells. JCell Biol.,70: 100a, 1976.

7. Gill, J. E., and Jotz, M. M. Further Observations on the Chemistry ofPararosaniline-Feulgen Staining. Histochemistry, 46. 147-160, 1976.

8. Gill, J. E., Jotz, M. M., Young, S. G., Modest, E. J.. and Sengupta. S. K.7-Amino-Actinomycin D as a Cytochemical Probe. I. Spectral Properties.J. Histochem. Cytochem., 23. 793-799, 1975.

9. Graham. R. M. The Cytologie Diagnosis of Cancer, Ed. 2. Philadelphia:W. B. SaundersCo.. 1963.

10. Graumann. W. Zur Standardisierung des Schiffschen Reagens. Z. wiss.Mikroskopie, 67: 225-226, 1953.

11. Kasten, F. H. Cytochemical Studies with Acridine Orange and theInfluence of Dye Contaminants in the Staining of Nucleic Acids. Intern.Rev. Cytol.. 2T: 141-198.1967.

12. Kopp, R. E., Lisa. J.. Mendelsohn, J., Permick, B.. Stone. H., andWohlers, R. Coherent Optical Processing of Cervical Cytology SamplesJ. Histochem. Cytochem., 24. 122-137, 1976.

13. Patten, S. F., Jr. Diagnostic Cytology of the Uterine Cervix. Baltimore:The Williams & Wilkins Co., 1969.

14. Sprenger, E., Sandritter. W., Naujoks. H., Hilgarth. M., Wagner. D., andVogt-Schaden, M. Routine Use of Flow-Through Photometric Prescreening in the Detection of Cervical Carcinoma. Acta Cytol., 21: 435-440,1977.

15. TrÃ¡ganos, F., Darzynkiewicz, Z., Sharpless, T., and Melamed, M. R.Simultaneous Staining of Ribonucleic and Deoxyribonucleic Acids inUnfixed Cells Using Acridine Orange in a Flow Cytofluorometric System.J. Histochem. Cytochem., 25: 46-56, 1977.

16. Umana, R., and Dounce, A. L. Relationship of Rat Liver Cathepsins toAutolytic Degradation of Proteins of the Liver Cell Nucleus duringIsolation. Exptl Cell Res.,35. 277-292, 1964.

17. Von Bertalanffy, L., Masin, M.. and Masin, F. A New and Rapid Methodfor Diagnosis of Vaginal and Cervical Cancer by Fluorescence Microscopy. Cancer, 77: 873-887, 1958

18. Wheeless, L. L., Jr., and Patten, S. F., Jr. Slit-Scan Cytofluorometry.

19.Acta Cytol.. 77: 333-339, 1973.Wheeless, L. L., Jr., Patten, S. F., Jr.. and Cambier, M. A. Slit-ScanCytofluorometry: Data Base for Automated Cytopathology Acta Cytol.,79. 460-464. 1975.Wheeless, L. L., Jr., Patten. S. F., Jr., and Onderdonk, M. A. QuantitativeComparison of Slide and Suspension Technique for Acridine OrangeStaining of Human Cells. Acta Cytol., 78: 8-12, 1974.Wied, G. L.. Messina, A. M., Meier. P., and Rosenthal, E. Deoxyribonu-cleic Acid Assessments on Feulgen-Stained Endometrial Cells, andComparison with Fluorometric Values on Acridine Orange StainedMaterial. Lab. Invest.. 14: 1494-1499. 1965.Zanker, V. Ãœberden Nachweis definierter reversibler Assoziate (reversible Polymerisate") des Acridinorange durch Absorptions- und Fluo-roeszenzmessungen in WÃ¤ssrigerLÃ¶sung.Z. Physik. Chem., 799: 225-258,1952.

23. Zar, J. H. Biostatistical Analysis, pp. 105-107. Englewood Cliffs. N. J.:Prentice Hall. Inc., 1974.

20

21

22




1978;38:1893-1898. Cancer Res James E. Gill, Leon L. Wheeless, Jr., Carol Hanna-Madden, et al. of Human Tumor Cell NucleiA Comparison of Acridine Orange and Feulgen Cytochemistry

Updated version

http://cancerres.aacrjournals.org/content/38/7/1893

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/38/7/1893To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



a comparison of acridine orange and feulgen cytochemistry...

Documents