Virchows Archiv B Cell Pathol (1992) 62:45-53
V'wchowsArchivB Cell Patlwlog.v IncludingMolecularPatl~dogy
9 Springer-Verlag 1992
Centrifugal separation of carcinoma or atypical cells in voided urine * Craig D. AIbright 1' ** and John K. Frost 2
1 Department of Pathology, Quantitative Cytopathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 2 Now deceased; formerly Head, Division of Cytopathology and Quantitative Cytopathology Laboratory, Department of Pathology, The Johns Hopkins University School of Medicine ReceivedNovember 2, 1991 / AcceptedJanuary 24, 1992
Summary. A simple density gradient method was used
to separate atypical and cancer cells from non-cancer cells in voided urine from patients with transitional cell atypia (moderate and grave atypia) and bladder cancer (squamous cell carcinoma and transitional cell carcinoma). Prior to cell separation, the Saccomanno preserved cells were dispersed by homogenization. After cell separation (5 min • 1400 rpm), atypical and cancer cells were enriched up to 20-fold. Also, most of the leucocytes (6898%) and squamous cells (47-82%) were absent from density gradient specimen fractions containing the largest percentages of atypical and cancer cells. Peak purity ranges of atypical or cancer cells from different sample classes showed a large degree of overlap. This permitted the pooling of density gradient fractions enriched for atypical or cancer cells, thus increasing the efficiency of the method. Also, following centrifugation, the Papanicolaou-stained specimen fractions showed less background staining than the unprocessed controls, and the cells retained diagnostic morphologic features. We infer that this method may be a useful, low-cost approach for the morphologic study of developing cancers, not only from the urinary bladder, but also from the respiratory tract. Key words: Urinary bladder- Carcinogenesis - Cytology
Density gradient centrifugation
Cancer and atypical preneoplastic epithelial cells may be detected cytologically in voided urine before the parent lesions are grossly apparent (Griese and Tweeddale * This work was supported in part by a grant awarded to J.K.F. from the William Penn Foundation ** Present address." Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, North Carolina, USA Offprint requests to: C.D. Albright, Room 347, Lineberger Comprehensive Cancer Center, Campus Box 7295, The University of North Carolina, Chapel Hill, NC 27599, USA
1978; Koss et al. 1985). However, the sensitivity of urinary cytology can vary widely (e.g., 42-100%) (Lewis et al. 1976) depending on the adequacy and cellular complexity of the specimen. The obscuration of cancer cells by the presence of other cells (e.g., red blood cells, leukocytes, squamous cells) can contribute to the under interpretation of early, low-grade tumors of the bladder (Geoffrey and Crabbe 1961; Murphy et al. 1984), and upper urinary tract (DeMay and Grathwohl 1985; Piscioli et al. 1983). Previous approaches for the isolation of cells in urine have included including membrane filtration (Bales 1981; Harris etal. 1971; Kapila et al. 1984; Murphy et al. 1984; Verma and Tiwari 1981), cytocentrifugation (Bales 1981; Kapila et al. 1984; Koss et al. 1989) and spontaneous sedimentation of cells directly onto glass slides (Bots et al. 1964; de Voogt et al. 1977; Rofe 1955). These approaches concentrate all cell types present, and thus permit the obscuration of atypical and cancer cells by other cells (Failde etal. 1963). Previous studies (Morse and Melamed 1974) suggested that the selective removal of leukocytes from urine cytologic specimens may permit objective cytomorphologic studies of the pathogenesis of bladder cancers. In a series of studies, we have documented the use of a Ficoll density gradient centrifugation method for the selective enrichment of alcohol-fixed atypical and cancer cells in sputum (Albright et al. 1986; Frost et al. 1979; Pressman et al. 1981). Using this approach most of the necrotic debris and leukocytes were removed from specimen fractions enriched for the majority of cancer cells. In the present study, we have described a modification of these methods, which permits the separation and enrichment of atypical or cancer cells in voided urine samples.
Materials and methods Urine cytologic specimens. Spontaneous voided urine specimens obtained from patients in The Johns Hopkins Hospital were the
46
and the entire Millipore filter was reviewed for the presence of atypical and cancer cells (Albright et al. 1986; Frost et al. 1979; Pressman et al. 1981).
1.18
1.16
A
Results
1.14
E E
Dispersal of cells 1.12
tO
1.10
1.08
1.06
0
-
i 5
.
.
.
.
,
.
.
.
.
10
Fraction Number
, 15
.
.
.
.
,
20
A BOTTOM
Fig. 1. Discontinuous density gradient profiles grouped according to specimen class. The plot shows the physical density of specimen fractions from density gradients used for samples from transitional cell atypia (open squares) and cancer specimens (closed circles). Once discrete homogeneous solutions are layered, diffusion effects may replace sharp boundaries with slightly blurred transition zones. - - o - Atypical; - - r cancer
source of cells for these experiments. A total of 15 specimens were studied, including: transitional cell carcinoma (4), squamous cell carcinoma (4), grave (3), and moderate (4) transitional cell atypia. After aliquots of the freshly voided, unfixed specimens were processed for routine diagnostic screening, each specimen was resuspended in an equal volume of Saccomanno preservative (2% Carbowax 1540, 50% ethyl alcohol) (Saccomanno et al. 1963). These preserved samples were the starting cell suspensions for the work described in this article.
Specimen preparation. Hemacytometer counts were performed to determine the cellularity of the preserved samples before blending. The cells were then dispersed in a 250-ml, semi-micro, Eberbach blender container at high speed for 3 min (Frost et al. 1979). Postblending hemacytometer counts were performed. The specimen cellularity was then correlated with the percentages of cell types present in Papanicolaou-stained pre- and post-blending aliquots of the same specimens. Centrifugal cell separation. Following homogenization, 1 ml of cells was layered onto a discontinuous density gradient of Ficoll (Ficoll 400, Pharmacia LKB Biotechnology, Piscataway, N.J.) (Fig. 1). Centrifugal cell separation was performed at room temperature for 5 min at 1400 rpm in a swinging bucket rotor (Sorvall; Du Pont, Wilmington, Del.) at a centrifugal force of 188.6 g at the sample-gradient interface. After centrifugation, the entire density gradient was collected in successive 0.5 ml fractions. The density of the gradient at a given specimen fraction was determined by measuring the refractive index in an Abbe refractometer (Bauch and Lomb, Rochester, N.Y.). These refractive index values were converted to density using a calibration series (0-50% w/v Ficoll) (Frost et al. 1979). Cytomorphologic analys&. The cellular contents of each gradient fraction were collected on a Millipore filter (25 mm, 8 p.m-pore size; Millipore, Bedford, Mass.) and the cells were stained with a modified Papanicolaou technique (Albright et al. 1990; Gill et al. 1974). Differential cell counts were performed on 100-200 noncancer cells in ten randomly selected areas totalling g 1.23 mm 2,
T h e m e a n cell c o n c e n t r a t i o n in the S a c c o m a n n o preserved s t a r t i n g s a m p l e s r a n g e d f r o m 6.2 x 104 to 9.8 x 105 cells/ml. Cell r e c o v e r y f o l l o w i n g h o m o g e n i z a t i o n v a r i e d f r o m 60.7% to 2 1 5 % , w i t h a m e a n cell r e c o v e r y o f 9 6 . 8 % . H o w e v e r , there was n o significant effect o f h o m o g e n i z a t i o n on either the t o t a l n u m b e r o f cells p e r m i l l i m e t e r (Table 1), o r o n the p e r c e n t a g e s o f cell t y p e s p r e s e n t ( T a b l e 2 ) w h e n c o m p a r e d to c o n t r o l (i.e., m a t c h e d , p r e - h o m o g e n i z a t i o n ) a l i q u o t s (Fig. 2). In o r d e r to d e t e r m i n e the effects o f t u m o r d e v e l o p m e n t stage (i.e., m o d e r a t e a t y p i a , g r a v e a t y p i a , cancer), we c o m p a r e d the c e l l u l a r i t y o f the s t a r t i n g samples. We f o u n d t h a t the c o n c e n t r a t i o n o f cells in the s t a r t i n g s a m ples was l o w e r in m o d e r a t e a n d g r a v e t r a n s i t i o n a l a t y p i a s a m p l e s t h a n in t r a n s i t i o n a l cell c a r c i n o m a (Table 1). In this series, the p e r c e n t a g e s o f the v a r i o u s cell types d i d
Table 1. The mean concentrations of cells in Saccomanno preserved voided urine samples before and after homogenization to disperse cellular aggregates; the differences between pre- and post-blending samples were not statistically significant Cancer class b
MOD GRA SQC TCC
Number of cells/ml (• 105)a Pre-blending
Post-blending
2.01 _ 1.10 0.62 _+0.37 8.52_+0.81 9.82 _ 7.60
1.44___0.72 0.42 _+0.18 1.76_+ 1.34 8.15 + 6.51
" Mean_ SD; freshly voided urine samples were resuspended in Saccomanno's preservative and then blended for 3 min at high speed (Saccomanno et al. 1963) b MOD, Moderate transitional cell atypia; GRA, grave transitional cell atypia; SQC, squamous cell carcinoma; TCC, transitional cell carcinoma
Table 2. Effects of homogenization on the percentage composition of cells in Saccomanno's preserved voided urine samples. After homogenization there was a small increase in the relative number leukocytes present, apparently due to the dispersal of cellular aggregates. This procedure had no statistically significant effect on the percentages of cell types present Cell type
Leucocytes Transitional cells Squamous cells Cancer - atypical cells
Percent cell type a Pre-blending
Post-blending
66.0___3.5 5.8 _ 2.5 28.2 ___3.4 0.3 _ 1.2
70.8 + 3.4 3.1 + 2.1 25.7 ___1.1 0.4 + 1.3
a Mean_+ SD based upon replicate cell counts performed in triplicate on the atypical and cancer samples using in this study
47
Fig. 2a, b. Dispersal of Saccomanno preserved cells in voided urine, a Unprocessed samples contain many ceils in cohesive dusters. b After Waring blending a significant proportion of the cells occur singly. Papanicolaou stained membrane filter, x 400
Table 3. Composition, purity and enrichment of Saccomanno preserved cells in voided urine samples Cancer class
Cell type a Leukocyte
Transitional
Squamous
Cancer
SQC
f----(90.0 + 10.5)% P=94.1% p = 1.065-+0.004 E = 1.0 x
f = (8.0 -+ 8.4)% P=49.6% p=1.111 • E = 6.2 x
f-- (1.9 -+ 3.5)% P=99.9% p = 1.157• E = 53.5 x
f = (0.07 + 0.05)% P = 1.4% p = 1.116• E = 20.4
TCC
f = (63.3 _+24.1)% P = 92.0% p = 1.069 +0.003 E=l.5x
f= (5.2 _+11.1)% P = 57.6% p = 1.088 +_0.031 E=ll.1 x
f = (31.3 -+23.3)% P = 100% p = 1.152• E=3.2x
f= (0.2 _+0.2)% P = 0.2% p = 1.146_+ 0.012 E=l.0x
GRA
f= (44.2 _ 32.2)% P =91.4% p = 1.069-+0.001 E=2.1x
f= (6.4_ 3.7)% P = 19.9% p = 1.139+0.037 E=3.1x
f= (50.9 + 32.4)% P - 100% p=1.135• E=2.0x
f= (0.5 +_0.7)% P = 0.7% p = 1.119-+0.017 E=1.3 x
MOD
f= (60.9-+ 24.4)% P = 8%9% p = 1.067 -+0.002 E = 1.4x
f= (3.9-+ 9.6)% P = t 1.4% p = 1.083 -+0.025 E = 2.9x
f = (34.6-+ 23.8)% P = 100% p = 1.130 -+0.025 E = 2.9x
f= (0.5 +_0.6)% P = 0.3% p = 1.129 -+0.012 E = 0.6x
a The mean percentages -+ SD of cells from voided urine samples before centrifugal cell separation are shown. After centrifugation and collection of sample gradient fractions, the relative frequencies (i.e., purities) of each cell type were determined for all specimen fractions. The peak purities (P) and densities (i.e., p +_SD) of the
individual peak purity fractions, and the peak enrichments (E = P/f) are shown for each cell type. For example, the 92% peak purity of leukocytes in transitional cell carcinoma samples if found at a density of 1.069 +0.003 g/ml, resulting in a 1.5-fold enrichment of leukocytes compared with the unprocessed starting samples
n o t v a r y significantly (Table 3). T h e r e f o r e the increase in the cellularity o f the c a n c e r s a m p l e s was d u e to a n increase in the p e r c e n t a g e s o f all cell t y p e s r a t h e r t h a n to a n increase in a specific cell t y p e (e.g., leukocytes).
Less t h a n 2 % o f the l e u k o c y t e s o r t r a n s i t i o n a l cells were r e c o v e r e d in the PPso r a n g e o f the a t y p i c a l cells. T h e PPso r a n g e o f the slight to m o d e r a t e l y a t y p i c a l cells (fractions 10-13; p = 1.115-1.129 g/ml) was s u b s t a n t i a l l y free o f d e b r i s a n d i n f l a m m a t o r y cells. H o w e v e r , the PPso r a n g e for n o r m a l s q u a m o u s cells s h o w e d a b r o a d distrib u t i o n , o v e r l a p p i n g c o n s i d e r a b l y w i t h the PPso r a n g e o f the a t y p i c a l cells. As s h o w n in Fig. 3, the m a x i m u m purities for l e u k o cytes, t r a n s i t i o n a l a n d s q u a m o u s cells were 8 8 % at p = 1 . 0 6 7 g / m l , 11% at p = l . 0 3 g/ml, a n d 100% a t p =
Moderate atypia samples F o l l o w i n g c e n t r i f u g a t i o n , the 5 0 % p e a k p u r i t y (PPso) ranges for leukocytes, s q u a m o u s a n d t r a n s i t i o n a l cells a n d a t y p i c a l cells were well s e p a r a t e d (Figs. 3 a n d 4).
48 a
b 100 -
go
m
12-
; 10-
80 -
a-
70 -"
60"
5o:
iD
4o: 0. 3o1
0-
4
D
2O -"
2
I0-" 0
9 ..,
.... 5
, .... 10
Fraction
, .... 15
~
0
20
. . . .
i
. . . .
b
. . . .
Fraction
Number
r
d
100
0.25
A
0.20
0.15
O L.,
50
O L.
~; 0.I0 a.
40
i
20
m
70 60
. . . .
Number
90 80
i
15
0
30 0.05 9
20 10 0
....
, .... 5
Fraction
,
....
10
, .... 15
Number
, 20
0.00 0
5
Fraction
1.168 g/ml, respectively9 The peak purity for the atypical transitional cells ( P = 0 . 3 % ) occurred at p = 1.129 g/ml, and was within the PPso range for squamous cells9 In Table 3 these peak purity values are compared to the percentages of the cell types in the Saccomanno preserved starting samples. The enrichment values for specific cell types correlated with the purity of that cell type in the starting samples (Table 3).
Grave atypia samples The PP5o ranges for the individual classes of cells (data not shown) were consistent with their locations in moderate atypia samples (Fig. 3). Compared to moderate atypia, the PPso ranges for the atypical cells in from the grave atypia specimen class spanned a wider range of fraction densities (fractions 7-12; p = 1.071-1.119 g/ ml). Thus, there was an increase in the proportion of the leukocytes recovered in the grave atypical cell PPso range (30% vs < 2 % ) compared to moderate atypia samples. However, the peak purity (p = 0.7%) of atypical cells in grave atypia samples occurred at a density (p = 1.119 +_0.017 g/ml) similar to that for atypical cells from the moderate atypia samples (Table 3). The PPso range for squamous cells extended to a higher density range compared with moderate atypia samples, resulting in a greater enrichment of atypical cells (1.3-fold vs ~ unity) in grave compared to moderate atypia samples (Table 3).
10
15
Fig. 3a-d. Centrifugal separation of (a) leukocytes, (b) normal transitional epithelial cells, (e) squamous cells, and (d) atypical, preneoplastic transitional cells in moderate transitional cell atypia samples. The individual graphs show the percent cell type in each of 20 sequential gradient fractions. In these studies the PPso range is defined as the contiguous range of specimen fractions containing 50% of a particular cell type and maximizing purity (Frost et al. 1979). The length of the solid black lines above the graphs indicates that the PPso ranges for leukocytes, squamous and transitional cells are well separated9 The PPso range for minimal to moderately atypical cells overlaps the squamous cell PPso range but is well separated from those of leukocytes and transitional cells
20
Number
Squamous cell carcinoma samples The peak purity ( P = 1 . 4 % at 1.116 g/ml) and the PPso range (p = 1.074-1.116 g/ml) for cancer and atypical cells (data not shown) were similar to those observed for atypical cells from non-cancer samples (see above). Although most of the cellular debris and leukocytes were recovered in the low density range (fractions 1-5; p = 1.065-1.069 g/ml), the PPso specimen fractions for cancer and atypical cells often contained secondarily formed aggregates (2-4 cells each) of leukocytes. A 20.4-fold average enrichment for cancer and atypical cells compared to unprocessed samples was found (Table 3). In squamous cell carcinoma samples, cancer and atypical cells occurrd more often in the PPso range for normal squamous cells than was observed in moderate and grave atypia, or in transitional cell carcinoma samples.
Transitional cell carcinoma samples Following centrifugation, the PPso ranges for leukocytes, normal transitional cells, atypical and cancer cells, and squamous cells were well separated (Fig. 5). Compared to samples of moderate and grave atypia, there was an increase in the purity of leukocytes in the cancer cell PP5o range. This was apparently due to the occurrence of fewer squamous cells in higher density fractions in cancer samples (e.g., p = 1.111-1.171 vs 1.146-1.169 g/
49
Fig. 4. Photomicrographs of cells in urine after centrifugal cell separation showing the peak purity fraction for (a) leukocytes,(b) transitional cells, (e) squamous cells, and (d) atypical transitional cells.
Gradient fractions enriched for atypical ceilsrarely contain necrotic cellular debris or leukocytes. Papanicolaou stained membrane filter, x 400
ml in grave vs cancer), rather than to an increase in the percentage of leukocytes recovered in the PP5 o range for transitional cell carcinoma cells (Fig. 4). Similarly, the PPso range for leukocytes (fractions 2-4) contained abundant debris but rarely contained cancer ceils.
cell carcinoma cells (Fig. 7). In this PPso range, the cancer cells were enriched 1.1-fold compared to matched, unprocessed samples (Table 4). Leukocytes and transitional ceils were maximally enriched in pooled region 1, similar to the values obtained without the pooling of fractions (see Table 3 vs 4).
Pooling of gradientfractions Discussion To determine if the number of specimen fractions collected could be reduced, while retaining the enrichment of cancer cells, the centrifugal separation of a series of transitional cell carcinoma samples (n=4) was repeated. After centrifugation, the specimen fractions were pooled prior to cytopreparation. Figure 6 shows that at least 50% of the leukocytes, normal transitional cells, and squamous cells were found outside of the pooled series of fractions containing the PPso range for transitional
Urinary cytology is an important, non-invasive method of detection, diagnosis and follow-up of urinary tract cancers. These studies show that non-cancer cells, and atypical and cancer cells in voided urine cell be successfully enriched using density gradient centrifugal cell separation. In a series of samples from patients with cytologically diagnosed moderate to grave atypia, or with frank cancer, a selective enrichment of leukocytes, tran-
50
Fig. 5a-d. Centrifugal separation of cell types present in transitional cell carcinoma samples, as described in the legend to Fig. 3. Note that while the PPso ranges (solid black lines) are well separated, the percentage of leukocytes in high density regions (e.g., p > 1.11 gm/ml, fractions 10-15) is increased compared to atypia samples (Fig. 3). A similar observation was made on sputum cancer samples (Albright and Frost 1986; Frost et al. 1979)
Fig. 6. Effects of pooling of specimen fractions on the separation of cell types from transitional cell carcinoma. After centrifugal separation, gradient fractions 1-5, 6-12, and 13-20 were pooled onto separate membrane filters and are represented in this figure by density ranges 1, 2, and 3. The height of the bars shows the purity (i.e., percent cell type) of leukocytes (white bars), transitional cells (cross-hatched bars) and squamous cell (black bars) in the series of pooled gradient fractions. The numbers above the bars show the percentage of a given cell type recovered in that fraction (e.g., 58%, 34%, and 8% of all of the leukocytes were recovered in density ranges 1, 2 and 3, respectively). The inset shows that sequential gradient fractions 6-12 (i.e., density range 2) contain the PPso range for cancer cells, which is well separated from the respective PPso ranges for leukocytes and transitional cells (density range 1)
sitional cells, squamous cells and cancer cells was achieved. This method also selectively separated cellular debris from most of the atypical and cancer cells, resulting in a reduction in background staining in the cancer cell fractions. The cancer cell peak purity fractions contained cells with readily identifiable morphologie features of preneoplastic epithelial cell atypia and cancer. Previous studies found that fewer absolute numbers of cells were present in low-grade ( ~ 10 z cells/ml) than in high-grade tumors ( ~ 10 s cells/ml), and that a significant proportion of cases (10-30%) contained fewer than 50-100 cancer cells (Beyer-Boon and Voorn-de Hollander 1978; Kern 1975; Minami et al. 1978). The occurrence of inflammation can increase the exfoliation of bladder epithelial cells (Melamed 1976), and may obscure the presence of atypical and cancer cells exfoliated from early bladder neoplasms. In addition, the effects of cell-derived products (e.g., acidic lysosomal enzymes) on the local urine microenvironment (e.g., pH, osmolarity, etc.) can adversely affect cellular morphology (Pearson et al. 1981 ; Trump and Berezesky 1987; Trump et al. 1989). These conditions can reduce the actual number of atypical or cancer cells suitable for diagnostic evaluation, thus potentially leading to an under interpretation of the parent neoplastic process, especially in cancer cases (Harris et al. 1971; Johnston 1964; Kapilla et al. 1984; Umiker 1964). In the data presented here unenriched starting samples from preneoplastic transitional cell atypias exhibited a lower range of cellularity than ones from bladder cancers (i.e., 104-105 VS 105-106 cells/ml). It has also been found here that the cancer samples, compared to the
51
Fig. 7a-tl. Light microscopic morphology of cells in pooled gradient fractions after centrifugal separation of transitional cell carcinoma samples, a Pre-centrifugation control, b Pooled fraction 1 containing the PPso range for leukocytes and transitional cells.
Table 4. Effects of pooling of gradient specimen fractions on the composition, purity and enrichment of ceils in voided urine samples from patients with transitional cell carcinoma (n=4)
e Fraction 2 containing the PPso range for cancer and atypical cells, d Fraction 3 containing mainly squamous cells, fewer atypical and cancer cells, and abundant necrotic debris
Cell type a Leukocyte
Transitional
Squamous
Cancer
f=(51.9 + 24.3)% P = 64.2% E = 1.2x
f=(6.5 + 3.4)% P = 30.7% E = 4.7x
f= (41.3_+27.5)% P = 40.7% E = 1.0x
f=(0.2+_0.1)% P = 0.26% E = 1.3x
a After centrifugation the gradient fractions were pooled as described in the Materials and methods section. The relative frequencies (i.e., purities) and peak enrichments were determined for each cell type, as described in Table 3. For example, the 64.2% peak purity of leukocytes occurs in pooled density gradient region I (see Fig. 6), resulting in a 1.2-fold enriehmen compared with unprocessed starting samples. The individual enrichment values reported in this table represent the average enrichment over the pooled series of specimen fractions defined for a given region of the density gradient
52
preneoplastic samples, contained a lower percentage of atypical and cancer cells, and, variably, a higher percentage of leukocytes. From this one may infer that depending on the tumor development stage, different biologic processes could be involved in the obscuration of atypical and cancer cells: (1) low specimen cellularity in the case of early, preneoplastic atypias, and (2) dilution of atypical and cancer cells by leukocytes in the case of frank cancers. Previous studies have documented the wide variability (40-100%) in the sensitivity of urinary cytology (Harving etal. 1989; Koss etal. 1989). A portion of this variability may be attributed to the methods used for sample collection. Diagnostic sensitivity is also affected by the tumor development stage (Murphy et al. 1984), by the presence ofpyuria (Hermansen et al. 1989), and by artifacts (e.g., necrosis) related to cancer therapy (Brannen etal. 1989; Bretton et al. 1989; Broghamer et al. 1989). Earlier studies (Esposti and Zajicek 1972; Frost 1976; Koss and Bartels 1975; Koss et al. 1985; Pearson et al. 1981) have cited the need for inexpensive methods able to increase the relative frequencies of preneoplastic and cancer cells in urine cytologic samples. Existing methods include cytocentrifugation (Bales 1981; Harris etal. 1971 ; Kapila et al. 1984) or membrane filtration (Nielsen et al. 1983; Pearson et al. 1981; Schwinn and Harris 1976; Verma and Tiwari 1981). In addition, Minami et al. (1978) described a density gradient technique for the separation of unfixed cells in urine. In a single case, these authors found that leukocytes and cancer cells were maximally enriched in the same gradient fraction. All of these methods serve to concentrate all cell types present (Frost et al. 1967), and, thus, have a limited ability to enrich for a specific cell type. In the present studies, we have used a centrifugal cell separation method to successfully enrich for populations of leukocytes, transitional cells, squamous cells, and atypical and cancer cells in voided urine. The peak enrichments for these individual cell populations occurred in different density gradient fractions. Reproducible patterns of cell separation and enrichment were achieved for a series of 15 specimens from patients with moderate to grave transitional cell atypia, or with bladder cancers. This represents a distinct improvement over previously described methods. In our study, the patterns of cell separation obtained with urine samples were similar to ones seen for cells in sputum (Albright et al. 1986; Frost et al. 1979; Pressman et al. 1981), but at a significant reduction in the duration of centrifugal separation (i.e., 5 min vs 2 h). Moreover, we have shown the feasibility of pooling specimen fractions containing the respective PP5o ranges for leukocytes, cancer cells, and squamous cells, while at the same time retaining the ability to enrich for cancer cells. This protocol is not time-consuming, does not require expensive equipment, and the atypical and cancer cells express readily interpretable morphologic features. We conclude that centrifugal separation and enrichment of cells from bladder cancers may complement existing methods (e.g., flow cytometry, image analysis, immuno-
cytochemistry) (Albright et al. 1989; Koss et al. 1989; Reedy et al. 1990a, b) which are will suited for the objective study of developing bladder cancer.
References Albright CD, Frost JK, Pressman NJ (1986) Centrifugal separation of cells in sputum from patients with undifferentiated carcinoma. Cytometry 7: 536-543 Albright CD, Hay R, Jones RT, Resau JH (1989) Discrimination of normal and transformed cells in vitro by cytologic and morphologic analysis. Cytotechnology 2:187-201 Albright CD, Keenan KP, Colombo K, Resau JH (1992) Identification of cells from the human tracheo-bronchus in culture. J Tissue Culture Methods 13:5-12 Bales CE (1981) A semi-quantitative method for separation of urine sediment for cytologic examination. Acta Cytol 25: 324-326 Beyer-Boon ME, Voorn-den Hollander MJA (1978) Cell yield obtained with various cytopreparatory techniques for urinary cytology. Acta Cytol 22: 589-594 Bots GRAM, Went LN, Schaberg A (1964) Results of a sedimentation technique for cytology of cerebrospinal fluid. Acta Cytol 8 : 234-241 Brannen W, Lucas TA, Mitchell WT Jr (1973) Accuracy of cytologic examination of urinary sediment in the detection of urothelial tumors. J Urol 109:483-485 Bretton PR, Herr KW, Kimmel M, Fair WR, Whitmore WF Jr, Melamed MR (1989) Flow cytometry as a predictor of response and progression in patients with superficial bladder cancer treated with Calmette-Guerin. J Urol 141:1332-1336 Broghamer WL, Packer JE, Harty JI, Gilkey CM (1989) Cytohistologic correlation of urothelial lesions secondary to photodynamic therapy. Acta Cytol 33:881-886 DeMay RM, Grathwohl MA (1985) Signet-ring (colloid) carcinoma of the urinary bladder. Cytologic histologic and ultrastructural findings in one case. Acta Cytol 29:132-136 Esposti PL, Zajicek J (1972) Grading of transitional cell neoplasms of the urinary bladder from smears of bladder washings. Acta Cytol 16:529-537 Failde M, Eckert WG, Patterson JN (1963) A comparison of a simple centrifuge method and the millipore filter technique in urinary cytology. Acta Cytol 7:199-206 Frost JK (1976) Cost-effectiveness considerations of new cellular preparations. In: Wied GL, Bahr GF, Bartels PH (eds) The automation of uterine cancer cytology. Tutorials in cytology. Chicago, pp 321-327 Frost JK, Gill GW, Hankins AC, LaCorte F J, Miller KA, Hollander DH (1967) Cytology filter preparations: Factors affecting their quality for the study of circulating cancer ceils in the blood. Acta Cytol 11 : 363-373 Frost JK, Pressman NJ, Albright CD, Gill GW, Vansickel MH (1979) Centrifugal separation of cells in sputum. J Histochem Cytochem 27: 7-13 Geoffrey J, Crabbe S (1961) Cytology of voided urine with special reference to "benign" papilloma and some of the problems encountered in the preparation of the smears. Acta Cytol 5 : 233-240 Gill GW, Frost JK, Miller KA (1974) A new formula for a halfoxidized hematoxylin solution that neither overstains for requires differentiation. Acta Cytol 18:30(~311 Griese LJ, Tweeddale DN (1978) Pre-clinical cytological diagnosis of bladder cancer. J Urol 120:51-56 Harris M J, Schwinn CP, Morrow JW, Gray RL, Browell BM (1971) Exfoliative cytology of the urinary bladder irrigation specimen. Acta Cytol 15:385-399 Harving N, Petersen SE, Melsen F, Wolf H (1989) Urinary cytology in the detection of bladder tumours. Scand J Urol Mephrol [Suppl] 125:127-130
53 Hermansen DK, Badalament RA, Fair WR, Kimmel M, Whitmore WF Jr, Melamed MR (1989) Detection of bladder carcinoma in females by flow cytometry and cytology. Cytometry 10:739742 Johnston WD (1964) Cytopathological correlation in tumors of the urinary bladder. Cancer 17:867-880 Kapila K, Wadhwa SN, Verma K (1984) Evaluation of cytology diagnosis in bladder neoplasms. Indian J Med Res 80:314-320 Kern WH (1975) The cytology of transitional cell carcinoma of the urinary bladder. Acta Cytol 19:420-428 Koss LG, Barrels PH (1975) Urinary cytology device capabilities and requirements. Anal Quant Cytol 2:420-428 Koss LG, Deitch R, Ramanathan R, Sherman AB (1985) Diagnostic value of cytology of voided urine. Acta Cytol 29:810-816 Koss LG, Wersto RP, Simmons DA, Deitch D, Hertz F, Freed SZ (1989) Predictive value of DNA measurements in bladder washings: comparison of flow cytometry, image cytophotometry and cytology in patients with a past history of urothelial tumors. Cancer 64:916--924 Lewis RW, Jackson AC Jr, Murphy WM, Leblanc GA, Meehan WL (1976) Cytology in the diagnosis and follow-up of transitional cell carcinoma of the urothelium. J Urol 116:43-46 Lipponen PK, Collan Y, Eskelinen M J, Pesonen E, Sotarauta M (1990) Volume corrected mitotic index (M/V index) in human bladder cancer: relation to histologic grade (WHO), clinical stage (UIC) and prognosis. Scand J Urol 24: 39-45 Melamed MR (1976) Introduction to cytology of urinary tract. In: Wied GL, Koss LG, Reagen JW (eds) Compendium on diagnostic cytology. Tutorials in cytology. Chicago, pp 405-412 Minami R, Yokota S, Teplitz RL (1978) Gradient separation of normal and malignant cells. II. Application to in vivo tumor diagnosis. Acta Cytol 22:584-588 Morse N, Melamed MR (1974) Differential counts of cell populations in urinary sediment smears from patients with primary epidermoid carcinoma of bladder. Acta Cytol 18 : 312-315 Murphy WM, Crabtree WN, Jukkola AF, Soloway MS (1981) The diagnostic value of urine versus bladder washing in patients with bladder cancer. J Urol 126:320-322 Murphy WM, Soloway MS, Jukkola AF, Crabtree WN, Ford KS (1984) Urinary cytology and bladder cancer. The cellular features of transitional cell neoplasms. Cancer 53 : 1555-1565
Nielsen ML, Fischer S, H6gsberg E, Therkelsen K (1983) Adhesives for retaining prefixed urothelial cells on slides after imprinting from cellulosic filters. Acta Cytol 27:371-375 Pearson JC, Kromhout L, King EB (1981) Evaluation of collection and preservation techniques for urinary cytology, Acta Cytol 25: 327-333 Piscioli F, Detassis C, Pella E, Pusiol T, Reich A, Luciani L (1983) Cytologic presentation of renal adenocarcinoma in urinary sediment. Acta Cytol 27:383-390 Pressman N J, Albright CD, Frost JK (1981) Centrifugal separation of cells in sputum specimens from patients with adenocarcinoma. Cytometry 1:260-264 Reedy EA, Colombo-Burke KL, Resau JH (1990a) Discrimination of cell types in primary transitional cell carcinoma by monoclonal anti-cytokeratin antibodies. Pathobiology 58: 304-311 Reedy E, Heatfield B, Trump B, Resau J (1990b) Correlation of cytokeratin patterns with histopathology during neoplastic progression in the rat urinary bladder. Pathobiology 58:15-27 Rofe P (1955) The cells of normal human urine. A quantitative and qualitative study using a new method of preparation. J Clin Pathol 8:25-31 Saccomanno G, Saunders RP, Ellis H, Archer VE, Wood BG, Beckler PA (1963) Concentration of carcinoma or atypical cells in sputum. Acta Cytol 7:305-310 Schwinn CP, Harris MJ (1976) Exfoliative cytology of the urinary bladder irrigation specimen. In: Wied GL, Koss LG, Reagan JW (eds) Compendium on diagnostic cytology. Tutorials in cytology. Chicago, pp 413-427 Trump BF, Berezesky IK (1987) The role of ion regulation in respiratory carcinogenesis. In: EM McDowell (ed) Lung carcinomas. Churchill Livingstone, New York, pp 162-174 Trump BF, Berezesky IK, Smith MW, Phelps PC, Elliget KA (1989) The relationship between cellular ion derequlation and acute and chronic toxicity. Toxicol Appl Pharmacol 97: 6-22 Umiker W (1964) Accuracy of cytologic diagnosis of cancer of the urinary tract. Acta Cytol 8:186-193 Verma K, Tiwari MC (1981) Millipore filter versus cytocentrifugation technique in cytopathological diagnosis. Indian J Med Res 74:135 140 Voogt HJ de, Rathert P, Beyer-Boon ME (1977) Preparatory techniques. In: Urinary cytology. Springer, Berlin Heidelberg New York, pp 7-13
V'wchowsArchivB Cell Patlwlog.v IncludingMolecularPatl~dogy
9 Springer-Verlag 1992
Centrifugal separation of carcinoma or atypical cells in voided urine * Craig D. AIbright 1' ** and John K. Frost 2
1 Department of Pathology, Quantitative Cytopathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 2 Now deceased; formerly Head, Division of Cytopathology and Quantitative Cytopathology Laboratory, Department of Pathology, The Johns Hopkins University School of Medicine ReceivedNovember 2, 1991 / AcceptedJanuary 24, 1992
Summary. A simple density gradient method was used
to separate atypical and cancer cells from non-cancer cells in voided urine from patients with transitional cell atypia (moderate and grave atypia) and bladder cancer (squamous cell carcinoma and transitional cell carcinoma). Prior to cell separation, the Saccomanno preserved cells were dispersed by homogenization. After cell separation (5 min • 1400 rpm), atypical and cancer cells were enriched up to 20-fold. Also, most of the leucocytes (6898%) and squamous cells (47-82%) were absent from density gradient specimen fractions containing the largest percentages of atypical and cancer cells. Peak purity ranges of atypical or cancer cells from different sample classes showed a large degree of overlap. This permitted the pooling of density gradient fractions enriched for atypical or cancer cells, thus increasing the efficiency of the method. Also, following centrifugation, the Papanicolaou-stained specimen fractions showed less background staining than the unprocessed controls, and the cells retained diagnostic morphologic features. We infer that this method may be a useful, low-cost approach for the morphologic study of developing cancers, not only from the urinary bladder, but also from the respiratory tract. Key words: Urinary bladder- Carcinogenesis - Cytology
Density gradient centrifugation
Cancer and atypical preneoplastic epithelial cells may be detected cytologically in voided urine before the parent lesions are grossly apparent (Griese and Tweeddale * This work was supported in part by a grant awarded to J.K.F. from the William Penn Foundation ** Present address." Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, North Carolina, USA Offprint requests to: C.D. Albright, Room 347, Lineberger Comprehensive Cancer Center, Campus Box 7295, The University of North Carolina, Chapel Hill, NC 27599, USA
1978; Koss et al. 1985). However, the sensitivity of urinary cytology can vary widely (e.g., 42-100%) (Lewis et al. 1976) depending on the adequacy and cellular complexity of the specimen. The obscuration of cancer cells by the presence of other cells (e.g., red blood cells, leukocytes, squamous cells) can contribute to the under interpretation of early, low-grade tumors of the bladder (Geoffrey and Crabbe 1961; Murphy et al. 1984), and upper urinary tract (DeMay and Grathwohl 1985; Piscioli et al. 1983). Previous approaches for the isolation of cells in urine have included including membrane filtration (Bales 1981; Harris etal. 1971; Kapila et al. 1984; Murphy et al. 1984; Verma and Tiwari 1981), cytocentrifugation (Bales 1981; Kapila et al. 1984; Koss et al. 1989) and spontaneous sedimentation of cells directly onto glass slides (Bots et al. 1964; de Voogt et al. 1977; Rofe 1955). These approaches concentrate all cell types present, and thus permit the obscuration of atypical and cancer cells by other cells (Failde etal. 1963). Previous studies (Morse and Melamed 1974) suggested that the selective removal of leukocytes from urine cytologic specimens may permit objective cytomorphologic studies of the pathogenesis of bladder cancers. In a series of studies, we have documented the use of a Ficoll density gradient centrifugation method for the selective enrichment of alcohol-fixed atypical and cancer cells in sputum (Albright et al. 1986; Frost et al. 1979; Pressman et al. 1981). Using this approach most of the necrotic debris and leukocytes were removed from specimen fractions enriched for the majority of cancer cells. In the present study, we have described a modification of these methods, which permits the separation and enrichment of atypical or cancer cells in voided urine samples.
Materials and methods Urine cytologic specimens. Spontaneous voided urine specimens obtained from patients in The Johns Hopkins Hospital were the
46
and the entire Millipore filter was reviewed for the presence of atypical and cancer cells (Albright et al. 1986; Frost et al. 1979; Pressman et al. 1981).
1.18
1.16
A
Results
1.14
E E
Dispersal of cells 1.12
tO
1.10
1.08
1.06
0
-
i 5
.
.
.
.
,
.
.
.
.
10
Fraction Number
, 15
.
.
.
.
,
20
A BOTTOM
Fig. 1. Discontinuous density gradient profiles grouped according to specimen class. The plot shows the physical density of specimen fractions from density gradients used for samples from transitional cell atypia (open squares) and cancer specimens (closed circles). Once discrete homogeneous solutions are layered, diffusion effects may replace sharp boundaries with slightly blurred transition zones. - - o - Atypical; - - r cancer
source of cells for these experiments. A total of 15 specimens were studied, including: transitional cell carcinoma (4), squamous cell carcinoma (4), grave (3), and moderate (4) transitional cell atypia. After aliquots of the freshly voided, unfixed specimens were processed for routine diagnostic screening, each specimen was resuspended in an equal volume of Saccomanno preservative (2% Carbowax 1540, 50% ethyl alcohol) (Saccomanno et al. 1963). These preserved samples were the starting cell suspensions for the work described in this article.
Specimen preparation. Hemacytometer counts were performed to determine the cellularity of the preserved samples before blending. The cells were then dispersed in a 250-ml, semi-micro, Eberbach blender container at high speed for 3 min (Frost et al. 1979). Postblending hemacytometer counts were performed. The specimen cellularity was then correlated with the percentages of cell types present in Papanicolaou-stained pre- and post-blending aliquots of the same specimens. Centrifugal cell separation. Following homogenization, 1 ml of cells was layered onto a discontinuous density gradient of Ficoll (Ficoll 400, Pharmacia LKB Biotechnology, Piscataway, N.J.) (Fig. 1). Centrifugal cell separation was performed at room temperature for 5 min at 1400 rpm in a swinging bucket rotor (Sorvall; Du Pont, Wilmington, Del.) at a centrifugal force of 188.6 g at the sample-gradient interface. After centrifugation, the entire density gradient was collected in successive 0.5 ml fractions. The density of the gradient at a given specimen fraction was determined by measuring the refractive index in an Abbe refractometer (Bauch and Lomb, Rochester, N.Y.). These refractive index values were converted to density using a calibration series (0-50% w/v Ficoll) (Frost et al. 1979). Cytomorphologic analys&. The cellular contents of each gradient fraction were collected on a Millipore filter (25 mm, 8 p.m-pore size; Millipore, Bedford, Mass.) and the cells were stained with a modified Papanicolaou technique (Albright et al. 1990; Gill et al. 1974). Differential cell counts were performed on 100-200 noncancer cells in ten randomly selected areas totalling g 1.23 mm 2,
T h e m e a n cell c o n c e n t r a t i o n in the S a c c o m a n n o preserved s t a r t i n g s a m p l e s r a n g e d f r o m 6.2 x 104 to 9.8 x 105 cells/ml. Cell r e c o v e r y f o l l o w i n g h o m o g e n i z a t i o n v a r i e d f r o m 60.7% to 2 1 5 % , w i t h a m e a n cell r e c o v e r y o f 9 6 . 8 % . H o w e v e r , there was n o significant effect o f h o m o g e n i z a t i o n on either the t o t a l n u m b e r o f cells p e r m i l l i m e t e r (Table 1), o r o n the p e r c e n t a g e s o f cell t y p e s p r e s e n t ( T a b l e 2 ) w h e n c o m p a r e d to c o n t r o l (i.e., m a t c h e d , p r e - h o m o g e n i z a t i o n ) a l i q u o t s (Fig. 2). In o r d e r to d e t e r m i n e the effects o f t u m o r d e v e l o p m e n t stage (i.e., m o d e r a t e a t y p i a , g r a v e a t y p i a , cancer), we c o m p a r e d the c e l l u l a r i t y o f the s t a r t i n g samples. We f o u n d t h a t the c o n c e n t r a t i o n o f cells in the s t a r t i n g s a m ples was l o w e r in m o d e r a t e a n d g r a v e t r a n s i t i o n a l a t y p i a s a m p l e s t h a n in t r a n s i t i o n a l cell c a r c i n o m a (Table 1). In this series, the p e r c e n t a g e s o f the v a r i o u s cell types d i d
Table 1. The mean concentrations of cells in Saccomanno preserved voided urine samples before and after homogenization to disperse cellular aggregates; the differences between pre- and post-blending samples were not statistically significant Cancer class b
MOD GRA SQC TCC
Number of cells/ml (• 105)a Pre-blending
Post-blending
2.01 _ 1.10 0.62 _+0.37 8.52_+0.81 9.82 _ 7.60
1.44___0.72 0.42 _+0.18 1.76_+ 1.34 8.15 + 6.51
" Mean_ SD; freshly voided urine samples were resuspended in Saccomanno's preservative and then blended for 3 min at high speed (Saccomanno et al. 1963) b MOD, Moderate transitional cell atypia; GRA, grave transitional cell atypia; SQC, squamous cell carcinoma; TCC, transitional cell carcinoma
Table 2. Effects of homogenization on the percentage composition of cells in Saccomanno's preserved voided urine samples. After homogenization there was a small increase in the relative number leukocytes present, apparently due to the dispersal of cellular aggregates. This procedure had no statistically significant effect on the percentages of cell types present Cell type
Leucocytes Transitional cells Squamous cells Cancer - atypical cells
Percent cell type a Pre-blending
Post-blending
66.0___3.5 5.8 _ 2.5 28.2 ___3.4 0.3 _ 1.2
70.8 + 3.4 3.1 + 2.1 25.7 ___1.1 0.4 + 1.3
a Mean_+ SD based upon replicate cell counts performed in triplicate on the atypical and cancer samples using in this study
47
Fig. 2a, b. Dispersal of Saccomanno preserved cells in voided urine, a Unprocessed samples contain many ceils in cohesive dusters. b After Waring blending a significant proportion of the cells occur singly. Papanicolaou stained membrane filter, x 400
Table 3. Composition, purity and enrichment of Saccomanno preserved cells in voided urine samples Cancer class
Cell type a Leukocyte
Transitional
Squamous
Cancer
SQC
f----(90.0 + 10.5)% P=94.1% p = 1.065-+0.004 E = 1.0 x
f = (8.0 -+ 8.4)% P=49.6% p=1.111 • E = 6.2 x
f-- (1.9 -+ 3.5)% P=99.9% p = 1.157• E = 53.5 x
f = (0.07 + 0.05)% P = 1.4% p = 1.116• E = 20.4
TCC
f = (63.3 _+24.1)% P = 92.0% p = 1.069 +0.003 E=l.5x
f= (5.2 _+11.1)% P = 57.6% p = 1.088 +_0.031 E=ll.1 x
f = (31.3 -+23.3)% P = 100% p = 1.152• E=3.2x
f= (0.2 _+0.2)% P = 0.2% p = 1.146_+ 0.012 E=l.0x
GRA
f= (44.2 _ 32.2)% P =91.4% p = 1.069-+0.001 E=2.1x
f= (6.4_ 3.7)% P = 19.9% p = 1.139+0.037 E=3.1x
f= (50.9 + 32.4)% P - 100% p=1.135• E=2.0x
f= (0.5 +_0.7)% P = 0.7% p = 1.119-+0.017 E=1.3 x
MOD
f= (60.9-+ 24.4)% P = 8%9% p = 1.067 -+0.002 E = 1.4x
f= (3.9-+ 9.6)% P = t 1.4% p = 1.083 -+0.025 E = 2.9x
f = (34.6-+ 23.8)% P = 100% p = 1.130 -+0.025 E = 2.9x
f= (0.5 +_0.6)% P = 0.3% p = 1.129 -+0.012 E = 0.6x
a The mean percentages -+ SD of cells from voided urine samples before centrifugal cell separation are shown. After centrifugation and collection of sample gradient fractions, the relative frequencies (i.e., purities) of each cell type were determined for all specimen fractions. The peak purities (P) and densities (i.e., p +_SD) of the
individual peak purity fractions, and the peak enrichments (E = P/f) are shown for each cell type. For example, the 92% peak purity of leukocytes in transitional cell carcinoma samples if found at a density of 1.069 +0.003 g/ml, resulting in a 1.5-fold enrichment of leukocytes compared with the unprocessed starting samples
n o t v a r y significantly (Table 3). T h e r e f o r e the increase in the cellularity o f the c a n c e r s a m p l e s was d u e to a n increase in the p e r c e n t a g e s o f all cell t y p e s r a t h e r t h a n to a n increase in a specific cell t y p e (e.g., leukocytes).
Less t h a n 2 % o f the l e u k o c y t e s o r t r a n s i t i o n a l cells were r e c o v e r e d in the PPso r a n g e o f the a t y p i c a l cells. T h e PPso r a n g e o f the slight to m o d e r a t e l y a t y p i c a l cells (fractions 10-13; p = 1.115-1.129 g/ml) was s u b s t a n t i a l l y free o f d e b r i s a n d i n f l a m m a t o r y cells. H o w e v e r , the PPso r a n g e for n o r m a l s q u a m o u s cells s h o w e d a b r o a d distrib u t i o n , o v e r l a p p i n g c o n s i d e r a b l y w i t h the PPso r a n g e o f the a t y p i c a l cells. As s h o w n in Fig. 3, the m a x i m u m purities for l e u k o cytes, t r a n s i t i o n a l a n d s q u a m o u s cells were 8 8 % at p = 1 . 0 6 7 g / m l , 11% at p = l . 0 3 g/ml, a n d 100% a t p =
Moderate atypia samples F o l l o w i n g c e n t r i f u g a t i o n , the 5 0 % p e a k p u r i t y (PPso) ranges for leukocytes, s q u a m o u s a n d t r a n s i t i o n a l cells a n d a t y p i c a l cells were well s e p a r a t e d (Figs. 3 a n d 4).
48 a
b 100 -
go
m
12-
; 10-
80 -
a-
70 -"
60"
5o:
iD
4o: 0. 3o1
0-
4
D
2O -"
2
I0-" 0
9 ..,
.... 5
, .... 10
Fraction
, .... 15
~
0
20
. . . .
i
. . . .
b
. . . .
Fraction
Number
r
d
100
0.25
A
0.20
0.15
O L.,
50
O L.
~; 0.I0 a.
40
i
20
m
70 60
. . . .
Number
90 80
i
15
0
30 0.05 9
20 10 0
....
, .... 5
Fraction
,
....
10
, .... 15
Number
, 20
0.00 0
5
Fraction
1.168 g/ml, respectively9 The peak purity for the atypical transitional cells ( P = 0 . 3 % ) occurred at p = 1.129 g/ml, and was within the PPso range for squamous cells9 In Table 3 these peak purity values are compared to the percentages of the cell types in the Saccomanno preserved starting samples. The enrichment values for specific cell types correlated with the purity of that cell type in the starting samples (Table 3).
Grave atypia samples The PP5o ranges for the individual classes of cells (data not shown) were consistent with their locations in moderate atypia samples (Fig. 3). Compared to moderate atypia, the PPso ranges for the atypical cells in from the grave atypia specimen class spanned a wider range of fraction densities (fractions 7-12; p = 1.071-1.119 g/ ml). Thus, there was an increase in the proportion of the leukocytes recovered in the grave atypical cell PPso range (30% vs < 2 % ) compared to moderate atypia samples. However, the peak purity (p = 0.7%) of atypical cells in grave atypia samples occurred at a density (p = 1.119 +_0.017 g/ml) similar to that for atypical cells from the moderate atypia samples (Table 3). The PPso range for squamous cells extended to a higher density range compared with moderate atypia samples, resulting in a greater enrichment of atypical cells (1.3-fold vs ~ unity) in grave compared to moderate atypia samples (Table 3).
10
15
Fig. 3a-d. Centrifugal separation of (a) leukocytes, (b) normal transitional epithelial cells, (e) squamous cells, and (d) atypical, preneoplastic transitional cells in moderate transitional cell atypia samples. The individual graphs show the percent cell type in each of 20 sequential gradient fractions. In these studies the PPso range is defined as the contiguous range of specimen fractions containing 50% of a particular cell type and maximizing purity (Frost et al. 1979). The length of the solid black lines above the graphs indicates that the PPso ranges for leukocytes, squamous and transitional cells are well separated9 The PPso range for minimal to moderately atypical cells overlaps the squamous cell PPso range but is well separated from those of leukocytes and transitional cells
20
Number
Squamous cell carcinoma samples The peak purity ( P = 1 . 4 % at 1.116 g/ml) and the PPso range (p = 1.074-1.116 g/ml) for cancer and atypical cells (data not shown) were similar to those observed for atypical cells from non-cancer samples (see above). Although most of the cellular debris and leukocytes were recovered in the low density range (fractions 1-5; p = 1.065-1.069 g/ml), the PPso specimen fractions for cancer and atypical cells often contained secondarily formed aggregates (2-4 cells each) of leukocytes. A 20.4-fold average enrichment for cancer and atypical cells compared to unprocessed samples was found (Table 3). In squamous cell carcinoma samples, cancer and atypical cells occurrd more often in the PPso range for normal squamous cells than was observed in moderate and grave atypia, or in transitional cell carcinoma samples.
Transitional cell carcinoma samples Following centrifugation, the PPso ranges for leukocytes, normal transitional cells, atypical and cancer cells, and squamous cells were well separated (Fig. 5). Compared to samples of moderate and grave atypia, there was an increase in the purity of leukocytes in the cancer cell PP5o range. This was apparently due to the occurrence of fewer squamous cells in higher density fractions in cancer samples (e.g., p = 1.111-1.171 vs 1.146-1.169 g/
49
Fig. 4. Photomicrographs of cells in urine after centrifugal cell separation showing the peak purity fraction for (a) leukocytes,(b) transitional cells, (e) squamous cells, and (d) atypical transitional cells.
Gradient fractions enriched for atypical ceilsrarely contain necrotic cellular debris or leukocytes. Papanicolaou stained membrane filter, x 400
ml in grave vs cancer), rather than to an increase in the percentage of leukocytes recovered in the PP5 o range for transitional cell carcinoma cells (Fig. 4). Similarly, the PPso range for leukocytes (fractions 2-4) contained abundant debris but rarely contained cancer ceils.
cell carcinoma cells (Fig. 7). In this PPso range, the cancer cells were enriched 1.1-fold compared to matched, unprocessed samples (Table 4). Leukocytes and transitional ceils were maximally enriched in pooled region 1, similar to the values obtained without the pooling of fractions (see Table 3 vs 4).
Pooling of gradientfractions Discussion To determine if the number of specimen fractions collected could be reduced, while retaining the enrichment of cancer cells, the centrifugal separation of a series of transitional cell carcinoma samples (n=4) was repeated. After centrifugation, the specimen fractions were pooled prior to cytopreparation. Figure 6 shows that at least 50% of the leukocytes, normal transitional cells, and squamous cells were found outside of the pooled series of fractions containing the PPso range for transitional
Urinary cytology is an important, non-invasive method of detection, diagnosis and follow-up of urinary tract cancers. These studies show that non-cancer cells, and atypical and cancer cells in voided urine cell be successfully enriched using density gradient centrifugal cell separation. In a series of samples from patients with cytologically diagnosed moderate to grave atypia, or with frank cancer, a selective enrichment of leukocytes, tran-
50
Fig. 5a-d. Centrifugal separation of cell types present in transitional cell carcinoma samples, as described in the legend to Fig. 3. Note that while the PPso ranges (solid black lines) are well separated, the percentage of leukocytes in high density regions (e.g., p > 1.11 gm/ml, fractions 10-15) is increased compared to atypia samples (Fig. 3). A similar observation was made on sputum cancer samples (Albright and Frost 1986; Frost et al. 1979)
Fig. 6. Effects of pooling of specimen fractions on the separation of cell types from transitional cell carcinoma. After centrifugal separation, gradient fractions 1-5, 6-12, and 13-20 were pooled onto separate membrane filters and are represented in this figure by density ranges 1, 2, and 3. The height of the bars shows the purity (i.e., percent cell type) of leukocytes (white bars), transitional cells (cross-hatched bars) and squamous cell (black bars) in the series of pooled gradient fractions. The numbers above the bars show the percentage of a given cell type recovered in that fraction (e.g., 58%, 34%, and 8% of all of the leukocytes were recovered in density ranges 1, 2 and 3, respectively). The inset shows that sequential gradient fractions 6-12 (i.e., density range 2) contain the PPso range for cancer cells, which is well separated from the respective PPso ranges for leukocytes and transitional cells (density range 1)
sitional cells, squamous cells and cancer cells was achieved. This method also selectively separated cellular debris from most of the atypical and cancer cells, resulting in a reduction in background staining in the cancer cell fractions. The cancer cell peak purity fractions contained cells with readily identifiable morphologie features of preneoplastic epithelial cell atypia and cancer. Previous studies found that fewer absolute numbers of cells were present in low-grade ( ~ 10 z cells/ml) than in high-grade tumors ( ~ 10 s cells/ml), and that a significant proportion of cases (10-30%) contained fewer than 50-100 cancer cells (Beyer-Boon and Voorn-de Hollander 1978; Kern 1975; Minami et al. 1978). The occurrence of inflammation can increase the exfoliation of bladder epithelial cells (Melamed 1976), and may obscure the presence of atypical and cancer cells exfoliated from early bladder neoplasms. In addition, the effects of cell-derived products (e.g., acidic lysosomal enzymes) on the local urine microenvironment (e.g., pH, osmolarity, etc.) can adversely affect cellular morphology (Pearson et al. 1981 ; Trump and Berezesky 1987; Trump et al. 1989). These conditions can reduce the actual number of atypical or cancer cells suitable for diagnostic evaluation, thus potentially leading to an under interpretation of the parent neoplastic process, especially in cancer cases (Harris et al. 1971; Johnston 1964; Kapilla et al. 1984; Umiker 1964). In the data presented here unenriched starting samples from preneoplastic transitional cell atypias exhibited a lower range of cellularity than ones from bladder cancers (i.e., 104-105 VS 105-106 cells/ml). It has also been found here that the cancer samples, compared to the
51
Fig. 7a-tl. Light microscopic morphology of cells in pooled gradient fractions after centrifugal separation of transitional cell carcinoma samples, a Pre-centrifugation control, b Pooled fraction 1 containing the PPso range for leukocytes and transitional cells.
Table 4. Effects of pooling of gradient specimen fractions on the composition, purity and enrichment of ceils in voided urine samples from patients with transitional cell carcinoma (n=4)
e Fraction 2 containing the PPso range for cancer and atypical cells, d Fraction 3 containing mainly squamous cells, fewer atypical and cancer cells, and abundant necrotic debris
Cell type a Leukocyte
Transitional
Squamous
Cancer
f=(51.9 + 24.3)% P = 64.2% E = 1.2x
f=(6.5 + 3.4)% P = 30.7% E = 4.7x
f= (41.3_+27.5)% P = 40.7% E = 1.0x
f=(0.2+_0.1)% P = 0.26% E = 1.3x
a After centrifugation the gradient fractions were pooled as described in the Materials and methods section. The relative frequencies (i.e., purities) and peak enrichments were determined for each cell type, as described in Table 3. For example, the 64.2% peak purity of leukocytes occurs in pooled density gradient region I (see Fig. 6), resulting in a 1.2-fold enriehmen compared with unprocessed starting samples. The individual enrichment values reported in this table represent the average enrichment over the pooled series of specimen fractions defined for a given region of the density gradient
52
preneoplastic samples, contained a lower percentage of atypical and cancer cells, and, variably, a higher percentage of leukocytes. From this one may infer that depending on the tumor development stage, different biologic processes could be involved in the obscuration of atypical and cancer cells: (1) low specimen cellularity in the case of early, preneoplastic atypias, and (2) dilution of atypical and cancer cells by leukocytes in the case of frank cancers. Previous studies have documented the wide variability (40-100%) in the sensitivity of urinary cytology (Harving etal. 1989; Koss etal. 1989). A portion of this variability may be attributed to the methods used for sample collection. Diagnostic sensitivity is also affected by the tumor development stage (Murphy et al. 1984), by the presence ofpyuria (Hermansen et al. 1989), and by artifacts (e.g., necrosis) related to cancer therapy (Brannen etal. 1989; Bretton et al. 1989; Broghamer et al. 1989). Earlier studies (Esposti and Zajicek 1972; Frost 1976; Koss and Bartels 1975; Koss et al. 1985; Pearson et al. 1981) have cited the need for inexpensive methods able to increase the relative frequencies of preneoplastic and cancer cells in urine cytologic samples. Existing methods include cytocentrifugation (Bales 1981; Harris etal. 1971 ; Kapila et al. 1984) or membrane filtration (Nielsen et al. 1983; Pearson et al. 1981; Schwinn and Harris 1976; Verma and Tiwari 1981). In addition, Minami et al. (1978) described a density gradient technique for the separation of unfixed cells in urine. In a single case, these authors found that leukocytes and cancer cells were maximally enriched in the same gradient fraction. All of these methods serve to concentrate all cell types present (Frost et al. 1967), and, thus, have a limited ability to enrich for a specific cell type. In the present studies, we have used a centrifugal cell separation method to successfully enrich for populations of leukocytes, transitional cells, squamous cells, and atypical and cancer cells in voided urine. The peak enrichments for these individual cell populations occurred in different density gradient fractions. Reproducible patterns of cell separation and enrichment were achieved for a series of 15 specimens from patients with moderate to grave transitional cell atypia, or with bladder cancers. This represents a distinct improvement over previously described methods. In our study, the patterns of cell separation obtained with urine samples were similar to ones seen for cells in sputum (Albright et al. 1986; Frost et al. 1979; Pressman et al. 1981), but at a significant reduction in the duration of centrifugal separation (i.e., 5 min vs 2 h). Moreover, we have shown the feasibility of pooling specimen fractions containing the respective PP5o ranges for leukocytes, cancer cells, and squamous cells, while at the same time retaining the ability to enrich for cancer cells. This protocol is not time-consuming, does not require expensive equipment, and the atypical and cancer cells express readily interpretable morphologic features. We conclude that centrifugal separation and enrichment of cells from bladder cancers may complement existing methods (e.g., flow cytometry, image analysis, immuno-
cytochemistry) (Albright et al. 1989; Koss et al. 1989; Reedy et al. 1990a, b) which are will suited for the objective study of developing bladder cancer.
References Albright CD, Frost JK, Pressman NJ (1986) Centrifugal separation of cells in sputum from patients with undifferentiated carcinoma. Cytometry 7: 536-543 Albright CD, Hay R, Jones RT, Resau JH (1989) Discrimination of normal and transformed cells in vitro by cytologic and morphologic analysis. Cytotechnology 2:187-201 Albright CD, Keenan KP, Colombo K, Resau JH (1992) Identification of cells from the human tracheo-bronchus in culture. J Tissue Culture Methods 13:5-12 Bales CE (1981) A semi-quantitative method for separation of urine sediment for cytologic examination. Acta Cytol 25: 324-326 Beyer-Boon ME, Voorn-den Hollander MJA (1978) Cell yield obtained with various cytopreparatory techniques for urinary cytology. Acta Cytol 22: 589-594 Bots GRAM, Went LN, Schaberg A (1964) Results of a sedimentation technique for cytology of cerebrospinal fluid. Acta Cytol 8 : 234-241 Brannen W, Lucas TA, Mitchell WT Jr (1973) Accuracy of cytologic examination of urinary sediment in the detection of urothelial tumors. J Urol 109:483-485 Bretton PR, Herr KW, Kimmel M, Fair WR, Whitmore WF Jr, Melamed MR (1989) Flow cytometry as a predictor of response and progression in patients with superficial bladder cancer treated with Calmette-Guerin. J Urol 141:1332-1336 Broghamer WL, Packer JE, Harty JI, Gilkey CM (1989) Cytohistologic correlation of urothelial lesions secondary to photodynamic therapy. Acta Cytol 33:881-886 DeMay RM, Grathwohl MA (1985) Signet-ring (colloid) carcinoma of the urinary bladder. Cytologic histologic and ultrastructural findings in one case. Acta Cytol 29:132-136 Esposti PL, Zajicek J (1972) Grading of transitional cell neoplasms of the urinary bladder from smears of bladder washings. Acta Cytol 16:529-537 Failde M, Eckert WG, Patterson JN (1963) A comparison of a simple centrifuge method and the millipore filter technique in urinary cytology. Acta Cytol 7:199-206 Frost JK (1976) Cost-effectiveness considerations of new cellular preparations. In: Wied GL, Bahr GF, Bartels PH (eds) The automation of uterine cancer cytology. Tutorials in cytology. Chicago, pp 321-327 Frost JK, Gill GW, Hankins AC, LaCorte F J, Miller KA, Hollander DH (1967) Cytology filter preparations: Factors affecting their quality for the study of circulating cancer ceils in the blood. Acta Cytol 11 : 363-373 Frost JK, Pressman NJ, Albright CD, Gill GW, Vansickel MH (1979) Centrifugal separation of cells in sputum. J Histochem Cytochem 27: 7-13 Geoffrey J, Crabbe S (1961) Cytology of voided urine with special reference to "benign" papilloma and some of the problems encountered in the preparation of the smears. Acta Cytol 5 : 233-240 Gill GW, Frost JK, Miller KA (1974) A new formula for a halfoxidized hematoxylin solution that neither overstains for requires differentiation. Acta Cytol 18:30(~311 Griese LJ, Tweeddale DN (1978) Pre-clinical cytological diagnosis of bladder cancer. J Urol 120:51-56 Harris M J, Schwinn CP, Morrow JW, Gray RL, Browell BM (1971) Exfoliative cytology of the urinary bladder irrigation specimen. Acta Cytol 15:385-399 Harving N, Petersen SE, Melsen F, Wolf H (1989) Urinary cytology in the detection of bladder tumours. Scand J Urol Mephrol [Suppl] 125:127-130
53 Hermansen DK, Badalament RA, Fair WR, Kimmel M, Whitmore WF Jr, Melamed MR (1989) Detection of bladder carcinoma in females by flow cytometry and cytology. Cytometry 10:739742 Johnston WD (1964) Cytopathological correlation in tumors of the urinary bladder. Cancer 17:867-880 Kapila K, Wadhwa SN, Verma K (1984) Evaluation of cytology diagnosis in bladder neoplasms. Indian J Med Res 80:314-320 Kern WH (1975) The cytology of transitional cell carcinoma of the urinary bladder. Acta Cytol 19:420-428 Koss LG, Barrels PH (1975) Urinary cytology device capabilities and requirements. Anal Quant Cytol 2:420-428 Koss LG, Deitch R, Ramanathan R, Sherman AB (1985) Diagnostic value of cytology of voided urine. Acta Cytol 29:810-816 Koss LG, Wersto RP, Simmons DA, Deitch D, Hertz F, Freed SZ (1989) Predictive value of DNA measurements in bladder washings: comparison of flow cytometry, image cytophotometry and cytology in patients with a past history of urothelial tumors. Cancer 64:916--924 Lewis RW, Jackson AC Jr, Murphy WM, Leblanc GA, Meehan WL (1976) Cytology in the diagnosis and follow-up of transitional cell carcinoma of the urothelium. J Urol 116:43-46 Lipponen PK, Collan Y, Eskelinen M J, Pesonen E, Sotarauta M (1990) Volume corrected mitotic index (M/V index) in human bladder cancer: relation to histologic grade (WHO), clinical stage (UIC) and prognosis. Scand J Urol 24: 39-45 Melamed MR (1976) Introduction to cytology of urinary tract. In: Wied GL, Koss LG, Reagen JW (eds) Compendium on diagnostic cytology. Tutorials in cytology. Chicago, pp 405-412 Minami R, Yokota S, Teplitz RL (1978) Gradient separation of normal and malignant cells. II. Application to in vivo tumor diagnosis. Acta Cytol 22:584-588 Morse N, Melamed MR (1974) Differential counts of cell populations in urinary sediment smears from patients with primary epidermoid carcinoma of bladder. Acta Cytol 18 : 312-315 Murphy WM, Crabtree WN, Jukkola AF, Soloway MS (1981) The diagnostic value of urine versus bladder washing in patients with bladder cancer. J Urol 126:320-322 Murphy WM, Soloway MS, Jukkola AF, Crabtree WN, Ford KS (1984) Urinary cytology and bladder cancer. The cellular features of transitional cell neoplasms. Cancer 53 : 1555-1565
Nielsen ML, Fischer S, H6gsberg E, Therkelsen K (1983) Adhesives for retaining prefixed urothelial cells on slides after imprinting from cellulosic filters. Acta Cytol 27:371-375 Pearson JC, Kromhout L, King EB (1981) Evaluation of collection and preservation techniques for urinary cytology, Acta Cytol 25: 327-333 Piscioli F, Detassis C, Pella E, Pusiol T, Reich A, Luciani L (1983) Cytologic presentation of renal adenocarcinoma in urinary sediment. Acta Cytol 27:383-390 Pressman N J, Albright CD, Frost JK (1981) Centrifugal separation of cells in sputum specimens from patients with adenocarcinoma. Cytometry 1:260-264 Reedy EA, Colombo-Burke KL, Resau JH (1990a) Discrimination of cell types in primary transitional cell carcinoma by monoclonal anti-cytokeratin antibodies. Pathobiology 58: 304-311 Reedy E, Heatfield B, Trump B, Resau J (1990b) Correlation of cytokeratin patterns with histopathology during neoplastic progression in the rat urinary bladder. Pathobiology 58:15-27 Rofe P (1955) The cells of normal human urine. A quantitative and qualitative study using a new method of preparation. J Clin Pathol 8:25-31 Saccomanno G, Saunders RP, Ellis H, Archer VE, Wood BG, Beckler PA (1963) Concentration of carcinoma or atypical cells in sputum. Acta Cytol 7:305-310 Schwinn CP, Harris MJ (1976) Exfoliative cytology of the urinary bladder irrigation specimen. In: Wied GL, Koss LG, Reagan JW (eds) Compendium on diagnostic cytology. Tutorials in cytology. Chicago, pp 413-427 Trump BF, Berezesky IK (1987) The role of ion regulation in respiratory carcinogenesis. In: EM McDowell (ed) Lung carcinomas. Churchill Livingstone, New York, pp 162-174 Trump BF, Berezesky IK, Smith MW, Phelps PC, Elliget KA (1989) The relationship between cellular ion derequlation and acute and chronic toxicity. Toxicol Appl Pharmacol 97: 6-22 Umiker W (1964) Accuracy of cytologic diagnosis of cancer of the urinary tract. Acta Cytol 8:186-193 Verma K, Tiwari MC (1981) Millipore filter versus cytocentrifugation technique in cytopathological diagnosis. Indian J Med Res 74:135 140 Voogt HJ de, Rathert P, Beyer-Boon ME (1977) Preparatory techniques. In: Urinary cytology. Springer, Berlin Heidelberg New York, pp 7-13
