Cytometry 12:614-621 (1991) 0 1991 Wiley-Liss, Inc.

An Improved Hedley Method for Preparation of Paraffin-Embedded Tissues for Flow Cytometric

Analysis of Ploidy and S-Phase' Thomas Heiden, Naining Wang, and Bernhard Tribukait2

Department of Medical Radiobiology, Karolinska Institute, S-10401 Stockholm, Sweden

Received for publication February 1, 1991; accepted April 17, 1991

A modification of the Hedley-method for flow cytometric DNA analysis of par- affin-embedded tissues is presented. De- waxed and dehydrated tissue from paraf- fin blocks was incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly without washing and centrifugation. The loss of material was minimized, which was ad- vantageous, for example, for the analysis of core-biopsies, and all measured sam- ples showed extremely low frequencies of clumped cell nuclei. This made it eas-

ier to detect polyploid nuclei and even rare nuclei of high ploidy could be iden- tified. S-phase analyses were more pre- cise, since the background originating from clumped debris particles was very low. The improved method was applied to the estimation of frequencies of high- polyploid nuclei found in various diploid, tetraploid, and aneuploid human myo- sarcomas of the uterus.

Keyterms: DNA flow cytometry, rare cells, core-biopsies

Flow cytometric DNA analysis of paraffin-embedded tissues was introduced by Hedley et al. in 1983 (8). Since then numerous studies which use this method have been published (see 9). The use of archival patho- logical material from patients whose clinical outcome is already known speeds the process of understanding the biological behavior of human tumors.

The flow cytometric analysis of archival tissue ac- cording to the Hedley-method has a reasonably high resolving power for estimations of ploidy. Owing to a higher debris background, compared to fresh material, and also a tendency to clumping of the cell nuclei, the analysis of S-phase cells and rare cell nuclei may, how- ever, be limited.

Some modifications of the basic Hedley-method (8) described in the literature were applied in this work: 100 pm sections were cut in order to minimize the por- tion of debris from cut nuclei (14). The sections were put into separate fine-mesh bags (14), which were cas- setted (1,131 and then placed in a tissue processor for automatic dewaxing and rehydrating (1) in order to simplify the procedure.

We also incubated the rehydrated tissue with sub- tilisin Carlsberg (Pronase, Sigma protease type XXIV), as was done by Jacobson et al. (10) and Amberson et al. (1). It has been observed in microscopic controls of the cell material after incubation that nuclei suspensions

with extremely low clumping frequencies were ob- tained. For this reason, in contrast to the preparations of the cited authors (1 and 101, the procedure described here omits all centrifugation steps. Centrifugation and washing not only cause loss of cell nuclei but can also produce considerable amounts of clumped cell nuclei and clumped debris. Thus, avoiding centrifugations, DNA-histograms with a reasonably high number of nu- clei were obtained even from small amounts of paraf- fin-embedded tissues such as those from core-biopsies. All histograms showed extremely small portions of clumped cell nuclei, and, in addition, a much lower exponential background. In this way, the analysis of S-phase and ploidy, including the analysis of rare nu- clei of high ploidy, is improved.

MATERIALS AND METHODS Preparation and Staining

The Hedley-method for DNA analysis of paraffin-em- bedded tissue (8) was applied with some modifications.

'This work was supported by the King Gustaf V:s Jubilee Founda- tion and the Heinrich Warner Foundation.

'Address reprint requests to Dr. Bernhard Tribukait, Department of Medical Radiobiology, Karolinska Institute, Box 60212, S-10401 Stockholm, Sweden.

IMPROVED DNA ANALYSIS TECHNIQUE 615

I I2S00 1

A

'361 '400 x=512 ' 2 c

I '200 '388 I

2c

FIG. 1. Typical DNA histograms of nuclei from paraffin-embedded mouse kidney treated with subtilisin Carlsberg and centrifuged (left) and not centrifuged (right) before analysis. X-axis, ploidy; y-axis,

Samples were prepared from paraffin-embedded mouse kidney and liver, and human testis carcinoma, myosar- comas of the uterus, normal lymphnodes, and surgical and core-biopsies of prostate carcinoma. Sections of 100 pm thickness were cut and put into fine-mesh bags (14) (one section per bag). Glass balls (1 mm diameter) were added before closing the bags in order to let the sections sink rather than float in solutions. A nylon gauze of 90 pm width was used to make the bags of about 1 cm x 1 cm size. The bags were placed into biopsy cassettes (Uni-cassette 4174, tissue-TER 111, Miles, Ames Divi- sion, Miles Laboratory, IN), which were put into a tis- sue processor (Shandon-Elliot). Dewaxing and rehy- drating were done by running the machine overnight. Two changes of xylene and two changes of ethanol in a sequence of 100, 95, 70, and 50% were made at room temperature. Each incubation lasted 1 h. The bags were then washed twice in distilled water for 20 min and were finally placed into 1 ml subtilisin Carlsberg solution for enzymatic digestion (0.1% Sigma protease XXIV, 0.1 M Tris, 0.07 M NaC1, pH 7.2) in a water bath at 37°C. The incubation time was 0.5 to 2 h depending on the type of tissue. The yield of cell nuclei in the solution was checked microscopically. After incubation without shaking, the samples were finally shaken for 20 min. The protease XXIV from Sigma Chemical Co., St. Louis, MO (E.C.3.4.21.14) is a crystallized enzyme with an activity of 7-14 units per mg solid. For com- parison, incubation with pepsin (0.5% pepsin 7180 from Merck, pH 1.5,30 min, 37°C) was used in samples from mouse kidney and liver, and human tumors.

Staining was carried out by adding directly 1 ml

number of nuclei. B shows the corresponding A histograms with an amplification of the y-scale (left: x 4, right: x 64). A 6c-clumping peak cannot be detected in the right histogram.

DAPI-phosphate solution (10 pM DAPI, 800 mM diso- dium-hydrogenphosphate) to the 1 ml subtilisin Carls- berg solution containing the bag and the suspended nuclei (final concentrations in the mixture: 5 pM DAPI, 0.05% subtilisin Carlsberg, 400 mM disodium- hydrogenphosphate, 0.05 M Tris, 0.045 M sodium chlo- ride, pH 8.2). Staining can also be done with 1 ml DAPI-citrate solution (10 pM DAPI, 800 mM trisodium citrate). It had been found previously that DAPI stain- ing at pH 8.8 resulted in narrow peaks of DNA histo- grams and that the maximal fluorescence intensity of DAPI did not change in the range between pH 7.0 and pH 9.5 (data not shown). Hence, a pH of 8.2 was ac- cepted for staining with DAPI. The upper limit of nu- cleus concentrations was at about 1 x lo6 nuclei per ml.

The samples were analyzed after a staining time of at least 1 h and up to 8 h at room temperature.

Tissue was also processed by magnetic stirring using small magnets of 1 mm diameter and 1.5 cm length. One millimeter of subtilisin Carlsberg solution con- taining the tissues was stirred in a water bath at 37°C for 15-45 min depending on the type of tissue. In this way also good yields of unclumped cell nuclei were ob- tained.

The concentrations of DAPI-stained nuclei in sus- pensions were determined using a Leitz epif luores- cence microscope and a Burker chamber.

The effect of centrifuging and washing the cell ma- terial after enzyme incubation was studied using two washing steps with distilled water and one more cen- trifugation before resuspending the pellet in DAPI so- lution according to Hedley et al. (8). The samples were

616 HEIDEN ET AL.

B 6 4 x

I I I I ’

I I 2 c 4 c

I 8 c

I 16c

FIG. 2. DNA histogram of nuclei from paraffin-embedded mouse liver. The tissue was treated as in Figure 1 (right). X-axis, ploidy; y-axis, number of cells. B shows the amplified A histogram ( x 64). A small portion of 16c nuclei can be recognized

centrifuged at 400g for 10 min and staining was carried out using 2 ml DAPI-solution (5 pM DAPI, 400 mM disodium-hydrogenphosphate, 0.05 M Tris, 0.045 M NaC1, pH 8.2).

Flow Cytometry The samples were analyzed with a PAS I1 flow cy-

tometer (Partec, Munster, BRD), which was equipped with a mercury arc lamp. The fluorochrome DAPI was excited in the ultraviolet (350-400 nm) and the fluo- rescence was measured in the blue region (>435 nm). The flow rate was normally 18 pl per minute. The fast- est flow rate, which was chosen for the analysis of some core biopsy samples with low concentrations of nuclei, was about ten times faster and resulted in slightly in- ferior CVs; the sample volume of 2 ml was then run in about 11 min. Usually at least 40,000 nuclei were an- alyzed from each sample at a measuring rate of about 500 nucleiisec. The 1024-channels-per-histogram op- tion of the acquisition program of the PAS I1 is espe- cially useful for the analysis of suspensions containing nuclei with a broad range of different DNA-contents. If, for example, diploid nuclei are measured in channel number 100, a broad range of higher ploidies can then be analyzed in the same histogram. The corresponding

G1 and G2 peaks can be easily correlated to each other using the linear x-scale.

The multicycle program for cell cycle analysis, writ- ten by P.S. Rabinovitch (Phoenix Flow Systems, San Diego, CA), which is also available from Partec (Mun- ster, BRD), was used for histogram analysis. This pro- gram provides the option of choosing between an expo- nential background fit, which is the general method of background analysis, and a background fit model which includes compensation for the effects of cutting of nuclei during sectioning of paraffin-embedded sam- ples. The magnitude of nuclear cutting is inferred from the region to the left of the diploid peak, corresponding compensations from all sources are applied to all other regions as well. This type of debris is added to the ex- ponential declining noise during least square fitting of the multicycle program.

RESULTS Comparison Between the Analysis of

Centrifuged and Uncentrifuged Samples Microscopic analysis of the uncentrifuged samples

after incubation with subtilisin Carlsberg showed “per- fect” suspensions of nuclei with extremely rare clump- ings, completely removed cytoplasm, good preservation

IMPROVED DNA ANALYSIS TECHNIQUE 617

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FIG. 3. DNA histogram of nuclei from a paraffin-embedded human myosarcoma of the uterus. The tissue was treated as in Figure 1 (right). X-axis, ploidy; y-axis, number of cells. B shows the amplified A histogram ( x 128). A small portion of nuclei can be recognized in

the 61 .6~ region. Since a small clumping peak around 45c contains less signals than one can find in the 61 .6~ region, the peak at 61 .6~ indicates the existence of true single nuclei in this region.

of nuclear details, and good stability independent of the incubation time. Suspensions from pepsin incubations showed poorer quality judged from higher clumping frequencies and lower stability with a longer incuba- tion time.

Figure 1 shows typical flow cytometric analyses of mouse kidney nuclei which had been treated with sub- tilisin Carlsberg. The cell nuclei shown in the left his- togram were centrifuged and washed prior to analysis, while those shown in the right histogram were directly stained without previous centrifugation. It was ob- served that the presence of the enzyme in unwashed samples did not increase the frequency of damaged nu- clei during a staining time of about 8 h at room tem- perature. Hence, inhibitors of the activity of subtilisin Carlsberg, such as aprotinin or alpha2-macroglobulin (4) were not added to the staining solution.

The histogram of washed and centrifuged nuclei (Fig. 1, left) shows a considerable amount of clumped cell nuclei: 2.6% events were measured in the 6c- clumping-peak. In contrast, in the uncentrifugated sample (Fig. 1, right) clumped nuclei are almost com- pletely absent: 0.01% events were found in the Gc-re- gion, and no 6c-peak can be distinguished.

The level of channel contents of the S-phase in the

left histogram is about the same as in the <2c-debris region. In the histogram of the uncentrifuged nuclei, those in S- or G2-phase can only be visualized with a higher magnification of the y-axis. A big difference can be seen in the levels of the channel contents between the S-phase region and the <2c-debris region.

It is obvious that the incubation of tissue from par- affin blocks with subtilisin Carlsberg yields nucleus suspensions with extremely low clumping frequencies. The portion of clumped nuclei and the exponentially declining debris background, originating from clumps among <2c-debris particles or from clumps among <2c-debris particles and G1-nuclei, is extremely small. This makes it feasible when avoiding centrifugation and washing to measure both nuclei in S-phase and rare events in the hypertetraploid region with a higher degree of accuracy. In the following sections the advan- tage of this improved DNA analysis is further exem- plified.

Detection of Rare Nuclei of High Ploidy Figures 2 and 3 are examples which demonstrate the

possibility of detecting even very small portions of cell nuclei of high ploidy. The 16c nuclei of mouse liver were analyzed at a frequency of 0.09% (Fig. 2). The

618 HEIDEN ET AL.

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FIG. 4. DNA histograms of nuclei from two paraffin-embedded human testis tumors which were treated as in Figure 1 (right). X-axis, ploidy; y-axis, number of cells. B shows the corresponding amplified A histograms (left: x 32, right: x 128). Small portions of aneuploid cells at 3 . 4 ~ (left) and 3c (right) can be recognized.

polyploid nuclei of a human myosarcoma of the uterus, the stem line of which was found at 7.7c, could be ob- served at a frequency of 0.1% in the 61 .6~ region (Fig. 3) .

Detection of Small Portions of Nuclei From Aneuploid Populations in the 2c4c region

The low debris-background in the S-phase region makes it easier to detect small quantities of nuclei from aneuploid populations within the S-phase region of dip- loid cells. Two examples from testis tumors are shown in Figure 4. The portions of the aneuploid populations are 3.5% in the left histogram and 0.2% in the right histogram. The existence of these aneuploid cell popu- lations was confirmed by additional measurements of other sections of tissue from the same patients.

S-Phase Estimations in Samples With Low Proliferation

Extremely low clumping frequencies result in a sig- nificantly lower level of channel contents in the S- phase region than in the <2c-debris region in samples with low proliferation (Figs. 1, 2,4, 5, and 6). Subtrac- tion of the exponentially fitted background then gives results which are obviously unreasonable. Using the subtraction of an exponentially fitted background in a histogram of nuclei from a normal human lymphnode (Fig. 5 ) , all events in the S- and G2-phase regions are subtracted (Fig. 6, middle).

Subtraction of the background, which has been fitted using the “sliced nuclei” option of the multicycle pro- gram of P.S. Rabinovitch (see Material and Methods), gives results which seem to be more reliable and indi- cate a small fraction of growing cells (Fig. 6, bottom), but the S- and G2-values could be regarded as maximal values.

DNA-analysis of Samples From Core-Biopsies By avoiding centrifugation and washing the loss of

cell material, which has been found to be about 85% using three centrifugations as it was done by Hedley et al. (8), is minimized. This is advantageous when sam- ples containing small amounts of tissue are prepared.

For example, of a total of 57 core-biopsies from hu- man prostate carcinomas only six samples could not be evaluated, while 51 gave a good quality histograms with a number of nuclei per histogram between 4,800 and 46,800 (mean value k s.d.: 25,700 * 9,600). Rea- sonable results for ploidy and also S-phase determina- tion could be obtained in most of these cases.

Application of the Method to the Analysis of High-Polyploid Nuclei From Human

Myosarcomas of the Uterus The role of so-called “giant-cells’’ in the diagnosis of

cancer and their possible prognostic significance have been discussed by several authors (2, 3, 6, 12, 15, 16). High-ploidy nuclei from paraffin-embedded tissue can

IMPROVED DNA ANALYSIS TECHNIQUE 619

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A

I '200 '400 x=!312 '

2 c

FIG. 5. DNA histogram of nuclei from a paraffin-embedded human normal lymphnode. The tissue was treated as in Figure 1 (right). X-axis, ploidy; y-axis, number of cells. B shows the amplified A histogram ( x 32).

not only be detected (see Figs. 2 and 3) but also ana- lyzed quantitatively using this technique.

Here, we studied the percentage of >8c nuclei in 66 samples from paraffin-embedded human myosarcomas of the uterus which all contained extremely low por- tions of clumped nuclei as judged from the lack of 6c- peaks. The difference in the proportions of high-ploidy cell nuclei between diploid, tetraploid, and aneuploid tumors is obvious (Fig. 7) with mean values 2 s.d. of 0.14% L 0.12% (n=8), 0.70% -t 0.51% (n= 15), 1.91% 2 2.0% (n = 45), respectively. Particularly among the aneuploid tumors, a wide range of >8c values was found which might be of value for further character- ization of the tumors.

DISCUSSION It is known that, compared to the preparations of

fresh cells for flow cytometric DNA analysis, prepara- tion of cells from paraffin-embedded tissues is more

time consuming, it yields higher CVs and more debris (9). The modification which is described in this paper does not reduce the amount of debris itself in samples from paraffin-embedded tissue, but due to reduction of clumping it reduces the debris background in the >2c- region considerably and hence makes it possible to an- alyze the cell cycle more precisely. Furthermore, the nearly entire absence of clumped cell nuclei makes it easier to detect polyploidisations, and even rare nuclei of high ploidy can be identified.

A good criterion for tetraploidy is, as has been em- phasized by Fossh and Thorud (7), and Joensuu and Kallioniemi (1 l), the presence of corresponding S- and G2,M-cells. The detection of these cells is easier when the method described is used, making the tumor clas- sification into the groups of diploid or tetraploid sam- ples more reliable.

It was found that the incubation of dewaxed and re- hydrated tissue from paraffin blocks with subtilisin

620 HEIDEN ET AL.

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FIG. 6 . The same histogram as in Figure 5 is shown with a loga- rithmic Y-scale. Three events of the total number of 117,000 nuclei can be recognized in the Gc-clumping region (top). Exponential back- ground subtraction removes all events in the s- and G2-region (ten- ter). Background subtraction according to P.S. Rabinovitch yields re- sults which indicate a small growing fraction (bottom).

Carlsberg results in nuclei suspensions with extremely low portions of clumpings. It is essential to omit all centrifugation and washing steps in order finally to obtain DNA-histograms with low clumping frequen- cies. Preparations of fresh tissue using ethanol fixation and pepsin incubation without centrifugation and washing before staining, as used by Zante et al. (17), which has been applied in our department to a large

15 8

6

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2

dipl. tetrapl. aneupl.

FIG. 7. Analysis of the portions of >Bc-cells in histograms of nuclei from 68 human myosarcomas of the uterus. The samples were treated as in Figure 1 (right). Diploid tumors, n = 8; tetraploid tumors, n = 15; aneuploid tumors, n = 43.

variety of different tumors, also reduced clumping sig- nificantly compared to preparations with centrifuga- tion. However, the use of subtilisin Carlsberg and the omission of all centrifugation steps in preparations of formalin fixed cell material resulted in the lowest ob- served clumping frequencies.

If, however, washing and centrifugation cannot be avoided, low concentrations of cell nuclei and low g- values are recommended.

Up to now, with the method described here, the fol- lowing human tumors have been prepared with good results: surgical biopsies and core-biopsies of normal prostate and prostate carcinoma, surgical biopsies of normal testis and testis carcinoma, myosarcoma of the uterus, carcinoma of the vulva, various lymphnode me- tastases, and normal lymphnodes. By avoiding centrif- ugation, the loss of cell material was minimized and the yield of nuclei was good even in samples containing only a small amount of tissue such as those from core- biopsies.

Some samples were stained with ethidium bromide instead of DAPI and also gave satisfactory results.

Incubation of samples which had been fixed in

IMPROVED DNA ANALYSIS TECHNIQUE 621

Bouin’s solution with subtilisin Carlsberg resulted in a poor quality of suspensions, just as reported by Hedley (9) for incubations with pepsin. While samples fixed in Bouin’s fluid gave poor results in all cases, the prepa- ration of formalin fixed archival material using the described subtilisin Carlsberg-protocol resulted in poor histograms only in some cases, for example, in 6 of 57 samples of core-biopsies from human prostate carcino- mas or in 12 of 78 samples from human myosarcomas of the uterus. The reason for the poor quality of some formalin fixed samples is probably inadequate fixation.

According to unpublished experiments, 0.1% subtili- sin Carlsberg solution lyses the cytoplasm and nucleus of 95% ethanol fixed cells within minutes. Cells which had been additionally fixed with formalin and then treated with 0.1% subtilisin Carlsberg only lost the cytoplasm while their nuclei remained intact. Subtili- sin Carlsberg obviously hydrolyzes cytoplasmatic pro- teins with high efficiency but not (all) nuclear proteins of formalin fixed cells, yielding suspensions of stable, bare nuclei. DNA-protein cross-linkages, which are formed by formalin fixation ( 5 ) , are eventually resis- tant to the action of subtilisin Carlsberg from Sigma (St. Louis, MO) as they are resistant to hydrolysis by pronase from Calbiochem (5). Hedley presumes that these nucleo-protein cross-linkages, which are formed by formalin fixation, are broken down by pepsin (8). This would perhaps explain the superior quality of sus- pensions from subtilisin Carlsberg incubations.

The applicability of the method has been demon- strated by the analysis of >&-nuclei of polyploid cells in samples from paraffin-embedded human myosarco- mas of the uterus. Such high-polyploid cells can be a marker of malignancy (2, 3, 6, 12, 15, 16) but their existence might also be of prognostic significance (2).

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