Sequential paraformaldehyde and methanol fixation for simultaneous ...

(9 1992 Wiley-Liss,

Inc.

Cytometry 13:432-444 (1992)

Sequential Paraformaldehyde and Methanol Fixation for Simultaneous Flow Cytometric Analysis of DNA, Cell Surface Proteins, and Intracellular Proteins' Agnese A. Pollice, J. Philip McCoy, Jr., Stanley E. Shackney,2 Charles A. Smith, Jyotsna Agarwal, Dennis R. Burholt, Laura E. Janocko, Francis J. Hornicek, Sarita G. Singh, and Robert J. Hartsock Allegheny-Singer Research Institute (A.A.P., S.E.S., C.A.S., J.A., D.R.B., L.E.J., F.J.H., S.G.S.) and Department of Pathology (R.J.H.), Allegheny General Hospital, and Departments of Pathology and Dermatology, The Pittsburgh Cancer Institute, University of Pittsburgh (J.P.M.), Pittsburgh, Pennsylvania Received for publication July 31, 1990; accepted October 15, 1991

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehydeimethanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone 0,< 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde 0, < 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde ef-

There are many published studies in which specific intracellular proteins have been studied quantitatively by means of flow cytometric immunofluorescent techniques (1,3-23,15-23,25), often in combination with cellular DNA content. Unfortunately, no clear consensus has emerged from these studies with regard to optimal cell fixation conditions for multiparameter flow cytometric analysis. In a number of studies, cells were fixed in methanol alone (8,15,19) or ethanol alone (1,3,7,9,10,16,18,25). However, comparative studies of alcohol with paraformaldehyde fixation have sug-

fects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37°C.Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors. 0 1992 Wiley-Liss, Inc. Key terms: Flow cytometry, cell fixation, membrane permeabilization, paraformaldehyde, methanol, multiparameter analysis, immunofluorescence

gested that there may be significant loss of cytoplasmic or nuclear proteins from cells following fixation with alcohol alone (17,181.

'This work was supported in part by Allegheny-Singer Research Institute Grant #90-049 (to S.E.S.) and by American Cancer Society Institutional Research Grant #IN 582 1 (to J.P.M.I. 'Address reprint requests to Stanley E. Shackney, Division of Medical Oncology, Allegheny General HospitaliAllegheny-Singer Research Institute, 320 E. North Avenue, Pittsburgh, PA 15212.

FIXATION METHOD FOR hfULTIPARBMETEI1 FCM ANA1,YSIS

Formaldehyde-based fixation procedures have varied widely among published studies. Formaldehyde or paraformaldehyde has been used for cell fixation, either with cell membrane permeabilization (46,ll-13,17,22), or without cell membrane permeabilization (21). Cell membrane permeabilization agents that have been used with formaldehyde fixation have included methanol (13), acetone (221, Triton X-100 (4,5,11,17,23), or lysolecithin (6). In the few studies that examined paraformaldehyde fixation conditions systematically, different conclusions were reached with regard to optimal fixation concentrations and fixation temperatures for the determination of intracellular antigens. Clevenger et al. (4) has recommended fixation with 0.5% paraformaldehyde at 4°C followed by cell membrane permeabilization with Triton X-100, while Mann et al. (17) have recommended fixation with 2% paraformaldehyde a t 37°C followed by cell membrane permeabilization. The fixation and membrane permeabilization procedures themselves may be associated with adverse effects on subsequent measurements. For example, variations in paraformaldehyde concentrations and long exposures to fixatives have been shown to alter propidium iodide DNA measurements (4). Additionally, detergents have been found to alter forward light scatter measurements (4,231. Cell permeabilization with lysolecithin results in extensive cell lysis, leading to low cell preparative yields (6). These effects would be of particular concern in multiparameter flow cytometry studies that include the simultaneous measurement of cell surface markers, intracellular proteins, and/or nuclear DNA. Because of these concerns; we have studied the effects of fixation with paraformaldehyde and methanol, separately and in combination, on intracellular and cell surface proteins by immunof luorescence and on concomitant cellular DNA measurements in several mammalian cell lines and on clinical samples of human tumors. The results of these studies are presented in this work.

MATERIALS AND METHODS Cell Lines GM 131 lymphoblasts obtained from the Human Genetic Mutant Cell Repository (Camden, NJ) were grown in suspension culture a t 37°C in a 5% CO, humidified atmosphere. The tissue culture medium consisted of RPMI 1640 medium supplemented with 20% viv heat-inactivated fetal bovine serum (HI-FBS), 25 mM HEPES buffer, 2 mM L-glutamine, and 30 pgiml gentamycin (GIBCO, Grand Island, NY). The tissue culture medium was changed twice a week. At confluence, cultures were split and subcultured at a 1:4 dilution. All tubulin studies were performed on cells that had been subcultured for 48 h. SKBR-3 cells, a human breast cancer cell line that expresses high levels of HER-2heu oncoprotein, were obtained from the American Type Culture Collection

433

IATCC, Rockville, MD) and were used as a HER-2ineuexpressing positive control in our flow cytometry studies. These cells were propagated in McCoy’s 5A medium containing 15% fetal calf serum and 0.3% gentamycin. SKBR-3 cells were split at confluence and subcultured a t a 1:4 dilution. CCRF-CEM and Molt4 (T lymphoblastic) cell lines and T-24 (bladder carcinoma) cell lines, obtained from ATCC, were maintained in RPMI 1640 medium supplemented with 10% viv HI-FBS and 30 pgiml gentamicin. The CCRF-CEM and Molt4 cells grew in suspension; the T-24 cells grew as monolayers and were harvested and subcultured using 0.025%>wiv trypsin in phosphate buffered saline (PBS). Media were changed twice weekly and the cells were assayed a t near conf luence. Normal peripheral blood was obtained by venipuncture from healthy volunteers or from patients with cutaneous T cell lymphomas who had leukemic T cells circulating in the peripheral blood and was used within 24 h of collection. Samples from patients with malignancy were obtained fresh and minced with scissors to obtain cells in suspension. Cell suspensions were filtered through 64 pm nylon mesh (Small Parts, Miami, FL) prior to fixation.

Reagents Purified grade paraformaldehyde and analytical grade methanol were purchased from Fisher Scientific Co. (Pittsburgh, PA). Bovine serum albumin, propidium iodide, Histopaque, and ribonuclease (RNase A, Type I; 1mg/ml, 90-100 unitsiml) were purchased from Sigma Chemical Co. (St. Louis, MO). RNase was heated for 10 min at 100°C to remove residual DNase activity. Antibodies All antibodies were titrated to determine saturating dilutions. Mouse monoclonal antitubulin antibody derived from clone #DM 1A was purchased from Sigma Chemical Co. (St. Louis, MO). This antibody was used at a n optimum saturating dilution of 1500. RAS 256 monoclonal antibody, a generous gift of Peter Hamer, NEN-Dupont, N. Billerica, MA, was used a t a 1:400 dilution. Monoclonal mouse anti-HER-2heu and polyclonal sheep anti-H ras antibodies, purchased from Cambridge Research Biochemicals (Valley Stream, NY), were used a t 1:1,000 dilutions. Antibodies to CD4 and CD15 were obtained from Becton Dickinson (Mountainview, CA) and were used at dilutions of 1: 100. Leukocyte common antigen was purchased from DAKO (Carpinteria, CA) and was used a t a dilution of 1:lOO. The specificity of each primary antibody was established by comparison with isotypic serum a s a primary antibody control. Second antibodies in indirect immunofluorescent studies included FITC-conjugated rabbit anti-mouse IgG, purchased from DAKO and were used a t a 1:20 dilution; Texas Red-conjugated rabbit anti-mouse IgG (Cappel Durham, NC) was used a t

434

POLLICE ET AL.

a 1 : l O O dilution; FITC-conjugated goat anti-mouse IgG (Organon Teknika-Cappel, West Chester, PA) was used a t a 1 5 0 dilution; FITC-conjugated rabbit anti-mouse IgG (AMAK, Westbrook, ME) was used a t a 1 5 0 dilution; and phycoerythrin-conjugated goat F (ab’), antimouse IgG(HL) (Tago, Inc., Burlingame, CAj was used a t a 1:lOO dilution.

Cell Preparation, Fixation, and Staining Peripheral blood mononuclear cells were isolated from whole blood of healthy donors on Histopaque (Sigma Chemical Co., St. Louis, MO) density gradients, and remaining erythrocytes were lysed with 0.9% NH,Cl. Exponentially growing GM 131cells in suspension cultures were harvested and washed in phosphate buffered saline (PBS) prior to staining a n d o r fixation. All cell centrifugations were performed a t 200 g. In experiments in which the cells were to be stained for surface marker proteins as well a s for cytoplasmic protein and DNA content, surface marker staining was performed prior to fixation using standard procedures for direct immunofluorescence (14). After staining of the surface antigen, the cells were washed in PBS, pelleted, and fixed sequentially with paraformaldehyde and methanol. After fixation, cells were washed and stained for intracellular antigen and DNA. Cells in suspension at a concentration of 1 x 106iml were fixed in freshly prepared paraformaldehyde alone at concentrations ranging from 0.1% to 10% wlv, fixed in methanol alone a t concentrations ranging from 22.5% to 70% vlv, or fixed sequentially with paraformaldehyde plus methanol. Paraformaldehyde fixation was performed with vortex agitation during the addition of fixative to prevent cell clumping. Fixation was performed at temperatures ranging from 4°C to 37”C, and the duration of cell exposure to paraformaldehyde ranged from 15 min to 2 h. Cells were then centrifuged and washed once with PBS. Methanol a t 4°C was then added while vortexing. Durations of exposure to methanol at 4°C ranging from 15 to 90 min were studied. The cells were then centrifuged and washed twice in PBS. Cell counts were obtained prior to fixation; cell viability was also determined by trypan blue exclusion prior to fixation. Cell counts were repeated after fixation in order to calculate cell yields. In studies employing detergents for cell permeabilization, paraformaldehyde-fixed cells were exposed to 0.1% Triton X-100 for two min a t 4°C. Intracellular DNA and protein staining were performed after the cells were washed free of the Triton X-100. In studies designed to determine the stability of intracellular protein and DNA fluorescence after fixation, cells were stored in PBS a t 4 T , and protected from light for varying time intervals between methanol fixation and staining. Intracellular antigens were stained using standard indirect and direct immunofluorescence techniques (13). DNA was stained with pro-

pidium iodide (PI) a t concentrations of 25-50 pg:’ml, in the presence of RNase.

Flow Cytometry Flow cytometric analysis were performed either on a FACStar cytometer (Becton Dickinson Immunocytometry, Inc., San Jose, CAI or on a n EPICS 753 cytometer (Coulter Cytometry, Hialeah, FL). The 488 nm line from a n argon laser was used for excitation in all experiments, and 530 nm band pass filters were used to collect the FITC fluorescence. Either a 590 nm long pass filter or 585 nm band pass filter was used to collect the PI fluorescence. All data were stored in list mode and analyzed either by the CONSORT 30 operating system (Becton Dickinson) or the EASY analysis system (Coulter). Relative Fluorescence Measurements of Intracellular Tubulin by Image Analysis In one set of experiments, image analysis studies and flow cytometric studies were performed in parallel on separate aliquots from cell suspensions that were fixed with various fixatives and stained with antitubulin antibody. Cells stained with a n unrelated isotypic first antibody and cells stained with second antibody alone served a s controls for specific antitubulin staining. Aliquots of cells in suspension were pipetted onto cleaned glass slides and coverslipped for image analysis. Phase contrast optics were used to identify individual cells. The light passing to the photomultiplier tube was restricted to that of each cell to be analyzed by framing with a n appropriately chosen aperture. Measurements of relative fluorescence intensity were made on randomly selected individual cells, using a Zeiss Photomicroscope I11 equipped with 100 watt Hg bulb, photomultiplier, fluorescein Zeiss filter combination (487717), and Intercolor 365019650 computer-controlled stage. For each cell, the background fluorescence of a n adjacent cell free region of comparable area was subtracted from the cellular fluorescence measurement. Measurements were made using a 40 x Neofluar objective. Relative cell fluorescence was expressed in arbitrary units. Over 50 cells were analyzed in each experimental group. Immunofluorescence Reference Standards and Quantitation of Antibody Binding For the tubulin studies, goat anti-mouse IgG-coated beads (Simply Cellular, Flow Cytometry Standards Corp., Research Triangle Park, NC) were used as a n external reference in order to minimize day-to-day variations in immunofluorescence due to differences in instrument performance and antibody staining. Goat anti-mouse IgG-coated beads were stained with mouse antitubulin followed by FITC conjugated rabbit antimouse antibodies, in parallel with the cell samples. Prior to each sample run, amplifier gain settings and photomultiplier tube voltage were adjusted to set the beads in a known reference channel. Antitubulin flu-

FIXATION METHOD FOR MULTIPARAMETER FCM ANALYSIS

435

FIG.1. Photomicrographs of GM 131 cells fixed in paraformaldehydeimethanol and stained for tubulin by indirect immunofluorescence. A. Interphase cell showing diffuse intracellular cytoplasmic staining; there is no nuclear staining. B. Anaphase cell showing dif-

fuse intracellular staining. There was no attempt to preserve microtubular structures during cell preparation. Anaphase chromosomes do not take up stain.

orescent antibody staining intensity could then be expressed quantitatively in relation to this reference, in arbitrary units. Total mean relative fluorescence intensitykell was calculated by dividing the total mean cellular fluorescence (minus mean fluorescence of unstained control) by the total mean stained bead fluorescence (minus mean fluorescence of unstained control beads). Mean nonspecific immunoglobulin fluorescence was calculated similarly on cells stained with second antibody only. Specific mean relative fluorescence per cell was calculated by subtracting mean nonspecific fluorescence from total mean fluorescence. Although the bead reference standard serves as a control for interexperimental variability in instrument performance characteristics, interexperimental variability in antitubulin antibody binding characteristics, and interexperimental variability in fluorescence intensity, it does not take into account such factors as intracellular exclusion of antibody, extracellular leakage, or denaturation of the target protein, or unavailability of the target protein antigenic sites to antibody due to extensive protein crosslinking. Thus, whereas the use of an external antibody-coated bead standard provides for interexperimental reproducibility of data collected at different times and run on the same instrument, it does not assure accurate representation of the total intracellular content of the cell constituent being measured.

RESULTS Optimal Cell Fixation Conditions for Immunofluorescence Studies of Intracellular Protein Cell fixation conditions for the detection of intracellular tubulin were investigated in GM 131 cells. The cells were fixed in paraformaldehyde alone, methanol alone, or sequential paraformaldehyde and methanol. Cell yields after fixation and cell staining exceeded 90 percent of the cells present in the prefixation samples with all three fixation procedures. Cell clumping was not a serious problem with any of the three fixation procedures; when present, cell clumping was seen most often in methanol-fixed samples. Cell morphology under phase contrast microscopy was well preserved with all three fixation procedures, although cells often appeared somewhat smaller after methanol fixation than after paraformaldehydelmethanol fixation at the same optical magnification. With all three fixatives, antitubulin immunofluorescence was appropriately observed in the cell cytoplasm, and was excluded from the cell nucleus (Fig. 1A).Anaphase chromosomes did not exhibit immunofluorescence (Fig. 1B). Since no attempt was made to preserve microtubules during cell preparation, cytoplasmic staining was diffuse, presumably reflecting antibody binding to free tubulin subunits. Mean intracellular antitubulin immunofluorescence

436 Table 1 Comparison of the Effects of Different Fixation Procedures on Intracellular Tubulin Irnmunofluorescence in GM 131 Cells"

I4O 120

T A

1

100

Fixative Paraformaldehyde alone Methanol alone Paraformaldehydei methanol Methanol/ uaraformaldehvde

Number of samples

Relative mean cell fluorescence (Arbitrary units) ? S.E.

7 7

0.7 2 0.2 1.5 f 0.4

11

4.5 2 0.6

4

1.0

* 0.05

ao 60

T

40

p < O.OOOZb p < 0.002

20

0

a < 0.006

"Exponentially growing GM 131 lymphoblasts were harvested, washed in PBS, and fixed in either 70% methanol, 2% paraformaldehyde followed by 70% methanol, or 70% methanol followed by 2% paraformaldehyde, a t cell concentrations of 10" cellsiml. Fixed cells were washed and stained with mouse antitubulin antibody and FITC-conjugated second antibody. Intracellular tubulin immunofluorescence was quantified by flow cytometry, using mouse immunoglobulin-coated beads as an external immunofluorescence reference standard. 'Students t-test vs. paraformaldehyde/methanol.

"1

T

50

B

40

30 20

was determined by flow cytometry following cell fixation by several different methods. Experiments were repeated several times to obtain multiple values for each data point; for each sample run, antibody-coated beads were used a s a n external standard (see Materials and Methods) and fluorescence signal amplifier gain settings were set in a fixed reference channel. Large, reproducible differences in the relative fluorescence of cells stained for tubulin were observed by flow cytometry, which were highly significant statistically (Student t test). These findings are summarized in Table 1. Fixation with paraformaldehyde followed by methanol produced over five times the antitubulin immunofluorescence than fixation with paraformaldehyde alone (p < 0.0002), and approximately three times the antitubulin immunofluorescence of methanol-fixed cells (p < 0.002). As shown in Table 1, cells fixed in paraformaldehyde followed by methanol exhibited almost five times the immunofluorescence of cells fixed with methanol followed by paraformaldehyde (p < 0.006). In a separate experiment, image analysis studies were performed in parallel with flow cytometry studies on aliquots of stained cells following fixation with paraformaldehyde alone, methanol alone, and paraformaldehyde followed by methanol. The results, shown in Figure 2 , confirm that tubulin-specific intracellular immunof luorescence is significantly greater following fixation with paraformaldehyde followed by methanol than after fixation with either paraformaldehyde alone or methanol alone (p < 0.0001). The same results were obtained both by image analysis (Fig. 2A) and by flow cytometry (Fig. 2B). We studied the effects of paraformaldehyde concentration, paraformaldehyde fixation temperature, and

10

0

MetOH

PF

PF

+ MetOH

FIG.2. A comparison of the effects of fixation method on intracellular antitubulin immunofluorescence: A by image analysis, and B by flow cytometry, on aliquots of the same cells. Dark bars represent isotype controls; bars with diagonal stripes represent controls stained with second antibody only; bars with vertical stripes represent cells stained with mouse antitubulin IgG and FITC-conjugated rabbit antimouse IgG. There was significantly greater specific cellular antitubulin immunofluorescence following paraformaldehyde!methanol fixation (PF+ MetOH) both by image analysis ( A ) and by flow cytometry (B) than after fixation with methanol alone (MetOH) or paraformaldehyde alone (PF).For further discussion, see text.

duration of exposure on the detection of intracellular proteins by immunofluorescence. In GM 131cells there was no significant effect of paraformaldehyde concentrations ranging from 0.25% to 4% (25"C, 1h exposure), on antitubulin immunofluorescence. Similarly, paraformaldehyde concentrations varying from 0.1% to 10% (25"C, 15 min exposure) had no significant effect on ras p21 immunofluorescence in T-24 bladder cancer cells. Because high paraformaldehyde concentrations may adversely affect light scatter properties and cellular DNA measurements, (see below) lower paraformaldehyde concentrations (0.25%)are preferred for routine use in multiparameter studies. The effects of paraformaldehyde fixation temperature on intracellular antitubulin immunofluorescence in GM 131 cells are shown in Table 2. Fixation with 2% paraformaldehyde for 1h at 37°C produces a relatively modest but statistically significant increase in antitubulin immunofluorescence above that observed a t

FIXATION METHOD FOR MULTIPARAMETER FCM ANALYSIS

Table 2 Effect o f Temperature on Intracellular Tubulin Determination in GM 131 Cells" Fixation temperature i"C1

4 37 25

Relative mean cell fluorescence Intensity (Arbitrarv units) i- S.E 2.84 * 0.08 4.29 i- 0.26 2.93 ? 0.22

p < 0.03

w < 0.03

"Exponentially growing GM 131 lymphoblasts were fixed at different temperatures in 2% paraformaldehyde for 1 h followed by 70% methanol. Intracellular tubulin immunofluorescence was quantitated by flow cytometry, using mouse immunoglobulin-coated beads as a n external reference standard. Six samples were analyzed at each time point.

lower temperatures. A similar fixation temperaturedependent increase in ras p21 immunofluorescence was observed in the T-24 bladder carcinoma cells (data not shown). Since high paraformaldehyde fixation temperature may adversely affect cellular DNA measurements (see below), there may be counterbalancing advantages to fixation with paraformaldehyde a t 25°C or lower for multiparameter studies that include DNA measurements. The duration of exposure to paraformaldehyde was evaluated over a range of 15 min t o 2 h. Once cells were fixed in paraformaldehyde at lor above room temperature for at least 15 min, subsequent storage at 4°C for up to 1week produced no significant loss of antitubulin immunofluorescence in GM 131 cells. Fixed clinical tumor samples have been stored for up to 2 months without significant loss of intracellular antitubulin immunof luorescence. The effects of methanol concentration and the duration of cell exposure to methanol were not found to be of critical importance, provided that methanol concentration exceeded 22.5%, and the duration of exposure was 1 h or longer. We routinely expose cells to 70% methanol for 1 h a t 4°C.

437

concentration of paraformaldehyde for preserving light scatter properties. At paraformaldehyde concentrations below 0.25%, the adverse effects of methanol fixation on light scatter properties predominate (Fig. 4A,B). At paraformaldehyde concentrations that exceed 0.25%)the paraformaldehyde itself would appear to degrade cell light scatter properties directly (compare Figure 4D,E with Fig. 4 0 .

Effects of Paraformaldehyde/methanolon Cell Surface Marker Studies Fixation-dependent effects on immunofluorescent cell surface markers were studied using monoclonal antibodies directed against lymphoid cell surface antigens (CD4) and monocyticigranulocytic (CD15) cell surface antigens. When stained cells fixed with 1% paraformaldehyde and methanol were compared with stained unfixed cells, there were no differences in staining intensity, and the proportions of cells expressing these cell determinants were comparable in fixed and unfixed cells.

Effects of Paraformaldehyde Fixation Conditions on Propidium Iodide Staining of DNA The fluorescence patterns of propidium iodide stained cells were found to be dependent on cell fixation conditions. With increasing paraformaldehyde concentration, there is a progressive decrease in propidium iodide staining intensity and a n increase in coefficient of variation (CV) of the Gall peak. The effects of paraformaldehyde concentration on propidium iodide staining of T-24 bladder carcinoma cells are shown in Table 3. When paraformaldehyde concentration exceeded 0.5%, the decrease in propidium iodide staining intensity and the increase in CV were quite pronounced. The temperature of paraformaldehyde fixation can also affect the intensity of propidium iodide staining, as shown in Table 4.Increasing fixation temperatures produced a decrease in Go,, peak channel number in T-24 bladder carcinoma cells. Effects of ParaformaldehydeimethanolFixation High concentrations of paraformaldehyde can proon Cell Light Scatter Properties duce spurious peaks in normal human peripheral blood The effects of various cell fixation procedures on for- leukocyte samples, as shown in Figure 5. At a paraward and right angle light scatter patterns of normal formaldehyde concentration of 0.25%)there was a shift peripheral blood leukocytes are shown in Figure 3. in Go,, peak position to a slightly lower channel in Paraformaldehydeimethanol fixation preserves normal comparison with the methanol-fixed diploid reference, light scatter properties sufficiently for gated discrimi- but no additional peaks were present (Fig. 5A). At a nation of leukocyte subsets (compare A and B in Fig. 3). paraformaldehyde concentration of 2%,the shift in Go,, Paraformaldehydeimethanol fixation is superior to peak position relative t o the methanol-fixed diploid refparaformaldehyde fixation with Triton X-100 cell per- erence was more pronounced, and a small, spurious meabilization in this regard (compare B and C in Fig. peak was observed to the right of the main Gail peak 3). Fixation with methanol alone results in severe deg- (Fig. 5B). When fresh cells were stained with antibody radation of light scatter properties, as shown in Figure to CD15 and fixed with 2% paraformaldehyde and 70% 3D. methanol, all of the C1)15-positive cells were included The effects of paraformaldehydeimethanol fixation in the peak with the higher DNA index; however, these on light scatter properties have been found to be de- cells represented only 15% of all the cells comprising pendent on paraformaldehyde Concentration. The data this peak. shown in Figure 4 suggest that there is a n optimal We have studied over 40 human tumor samples that

438

POLLICE ET AL.

2501

A

1

B

200

a, 250 -

C

n

c 200

Q Y

.., .

0

50

100

r . .

. _ ^ . ... . %:

,

:

~

..... ..I,..

150

.

,

1

_.. . ,

200

250

0

50

100

150

260

250

Forward Light Scatter FIG.3. A comparison of the effects of cell fixation procedures on light scatter patterns of normal human peripheral white blood cells. Forward and right angle light scatter pattern of: A, unfixed cells; B, cclls fixed with 0.254 w/v paraformaldehyde a t 4°C for 15 min, plus

22.5% methanol at 4°C for 1h; C, 0.25% w/v paraformaldehyde at 4'C for 15 min, plus 0.1% Triton X-100 for 2 min a t 4"C, and D, 50% methanol at 4°C for 1 h.

were fixed both in methanol alone and in paraformaldehyde (0.25% or 2%)imethanol. Aneuploid peaks that were identified in methanol-fixed cells were also present in paraformaldehydeimethanol-fixed samples. However, in paraformaldehyde/methanol-fixed tumor samples, the Go,, peaks of diploid cells and, when present, the Go,, peaks of aneuploid cells were both shifted to lower values than those of the corresponding peaks of methanol-fixed cells (Table 5 ) . As indicated in Table 5, the DNA indices of diploid cells present in methanol-fixed tumor samples corresponded closely to those of methanol-fixed normal lymphocytes that were used as a n external reference standard, with a mean DNA index of 0.99 f 0.02; diploid cells from the same tumor samples fixed in paraformaldehydeimethanol exhibited a mean DNA index of 0.73 k 0.21 relative to methanol-fixed normal lymphocytes (P = 0.0005). Aneuploid Go,, peaks in methanol-fixed samples exhibited a mean DNA index of 1.76 ? 0.73. In every case, the aneuploid peak in paraformaldehydeimethanolfixed cells from the same sample exhibited a lower DNA index than the aneuploid peak of the corresponding methanol-fixed cells, relative to methanol-fixed normal lymphocytes, with a mean DNA index of 1.18 2 0.64 (P = 0.001). The paraformaldehyde-induced decrease in propidium iodide fluorescence was greater for diploid cells

than for aneuploid cells within the same sample, as indicated in Table 6. In Table 6, methanol-fixed diploid cells in tumor samples are compared with a methanolfixed external normal lymphocyte standard, and paraformaldehyde/methanol-fixed diploid cells in tumor samples are compared with paraformaldehydeimethanol-fixed normal lymphocytes. The paraformaldehydei methanol-fixed normal lymphocytes and the paraformaldehyde/methanol-fixed diploid cells in the tumor samples exhibited comparable decreases in propidium iodide fluorescence; hence, the paraformaldehydei methanol-fixed diploid cells in the tumor samples exhibited a DNA index of 1.02 ? 0.10. In the methanolfixed samples, when the aneuploid cells in each sample were compared with the corresponding diploid cells in the same sample, their mean DNA index was 1.53 2 0.62. When the paraformaldehydeimethanol-fixedaneuploid cells were compared with their internal diploid reference cells, their DNA indices appeared higher than the DNA indices of their methanol-fixed aneuploid counterparts, with a mean DNA index of 1.77 k 0.82 ( p < 0.04). This is attributable to the relative insensitivity of paraformaldehydeimethanol-fixed aneuploid cells to the fixative-induced loss in propidium iodide fluorescence in comparison with the diploid cells in the same samples. Indeed, when the tumor cell samples are divided into those with aneuploid cells having

439

FIXATION METHOD FOR MULTIPARAMETER FCM ANALYSIS

B

A 200 7501 150 I

a,

100

w r

m 0

v)

50

2

0 0

50

0

100 150 200 250

50

100

150 200 250

250

E

C

50

0

50

.

100 150 200 250 0

.

50

100

150 200 2500

50

100 150 200 250

Forward Light Scatter FIG.4. The effects of paraformaldehyde concentration on cell light scatter properties of normal human peripheral blood leukocytes. Forward and right angle scatter of cells exposed to: A, 70% methanol only; B, 0.1% paraformaldehyde + 70% methanol; C, 0.25%

paraformaldehyde + 70% methanol, and D, 0.5%paraformaldehyde + 70%methanol E. 1%paraformaldehyde + 70% methanol For discussion, see text.

Table 3

formaldehydeimethanol-fixed samples, but the differences were not statistically significant (Table 8). Paraformaldehyde-fixed tumor samples may demonstrate additional aneuploid peaks in the hyperdiploid region that are not observed in corresponding methanol-fixed samples. At least in some instances, the apparent aneuploidy that is seen only in the paraformaldehyde-fixed cells may represent differential staining of abnormal cell subpopulations that would otherwise stain as diploid after methanol fixation but are biologically distinct from other presumably normal diploid cells that are present in the paraformaldehyde-fixed sample. This is illustrated by the example shown in Figures 6-8. The DNA histogram of methanol-fixed, propidium iodide stained cells obtained from a human breast cancer is shown in Figure 6A. There is a prominent diploid Go,,,peak in channel 40. A prominent near-tetraploid peak is also present, as well as a small hyperdiploid peak (Fig. 6A, arrow). The corresponding DNA histogram obtained with paraformaldehydeimethanol-fixed cells is shown in Figure 6B. The diploid Go,, peak appears less prominent than in the methanol-fixed cell sample; the prominent near-tetraploid Go,l peak is readily identified. The hyperdiploid (Fig. 6B, arrow) peak is shifted to higher channels than in the methanol-fixed cells, appears more prominent than in the methanol-fixed cells, and exhibits a clear notch (compare with Fig. 6A). These findings would suggest that there is a subpopulation of cells that appears to be dip-

Effects of Paraformaldehyde Concentration on Propidium Iodide Staining of Cellular DNA in T-24 Bladder Cancer Cells" Paraformaldehyde concentration (% wiv) 0 0.1 0.25

0.5 1

Go,, peak channel number

cv (96)

46 45 42 41 31

4.4 3.1 2.4 2.4 7.2

"Cells fixed in paraformaldehyde at 4°C for 15 min, followed by 22.5%methanol fixation.

DNA indices less than or equal to 1.5 after methanolfixation, and those with aneuploid cells having DNA indices greater than or equal to 1.5, it is apparent that i t is the cells with DNA indices greater than triploid that exhibit the greatest relative resistance to paraformaldehyde-induced loss of propidium iodide fluorescence (Table 6). Coefficients of variation of the position of the Go,, peak were comparable for methanol-fixed and paraformaldehydeimethanol-fixed normal lymphocytes, but were higher in paraformaldehydeimethanolfixed apparently diploid cells in tumor samples than in methanol-fixed diploid cells in tumor samples (Table 7). S-phase cell fractions tended to be higher in para-

440

POLLICE ET AL.

Table 4 Effects of Paraformaldehyde Fixation Temperature on Propidium Iodide Staining of Cellular DNA in T-24 Bladder Cancer Cells" Temperature

cv ('70)

4

Go,, peak channel number 70

25 37

61 51

2.6 4.3

("C)

2.3

"Cells fixed in 0.5% paraformaldehyde for 15 min, followed by 22.5% methanol fixation.

A

v 240

DNA Content FIG.5. The effects of paraformaldehyde concentration on the position of the Go/l peak relative to that of a n alcohol-fixed diploid reference. A. Sample fixed in 0.25% paraformaldehyde/methanol and stained with propidium iodide. Diploid reference cells were fixed in 70% methanol and set in channel 60 (vertical line). The Go,, peak position of paraformaldehyde/methanol fixed cells had a modal value in channel 49 (CV, 3.6%). B. Sample fixed in 2% paraformaldehyde/ methanol and stained with propidium iodide. The methanol-fixed diploid reference was set in channel 60 (vertical line). The Gotl peak position of the paraformaldehydeimethanol-fixed cells was in channel 24 (CV, 3.6470).There was a small, spurious peak to the right of the main Go), peak (modal value 31). For discussion, see text.

loid with methanol fixation but is stained differently from other diploid cells by propidium iodide after paraformaldehydelmethanol fixation. Since the paraformaldehydeimethanol-fixed cells were stained simultaneously for DNA, intracellular HER-Pheu oncogene protein product (Fig. 6 0 , and intracellular ras p21 protein (Fig. 6D),the notched hyperdiploid peak could be characterized further by multiparameter flow cytometric analysis. It is apparent from Figure 6C that many cells contained as little HER-2ineu as the normal lymphocyte reference (ver-

tical dashed line with minus sign); there were also cells that expressed larger amounts of HER-2ineu protein than in the normal lymphocytes, including some that contained as much as the HER-B/neu protein-positive reference cells (SKBR-3 cell line; vertical dashed line with plus sign, Fig. 6C).Similarly, Figure 6D shows t h a t there were many cells that expressed a n amount of ras p21 protein that was comparable to that of normal lymphocytes (vertical dashed line with plus sign), a s well a s cells with increased amounts of ras p21 protein, some of which were comparable t o those seen in SKBR3 cells (vertical dashed line with plus sign). We examined gated subsets of cells that were derived from each of the three bivariate distributions that were generated from our multiparameter study. Figure 7 shows the bivariate distributions and gate contours that were found to be most relevant to the characterization of the hyperdiploid peak shown in Figure 6B (arrow). Population I was gated on hyperdiploidy and low ras p21 protein expression (Fig. 7A). Population I1 was gated on hyperdiploidy and increased ras p21 protein expression (Fig. 7B). Population 111 was gated on low ras p21 expression and low HER-2lneu expression (Fig. 7C). It is apparent from Figure 8 that the notched hyperdiploid peak of Figure 6B actually consists of two hyperdiploid peaks; one contained cells with increased ras p21 expression and, the other contained low ras p21 cells. Population I, consisting of hyperdiploid, low ras p21 cells stained slightly more intensely with propidium iodide, and occupied a position to the right of the notch in the hyperdiploid peak (Fig. SA).Population 11, which consisted of hyperdiploid, high ras p21 cells, stained less intensely with propidium iodide, and occupied a position to the left of the notch (Fig. 8B). In confirmation of this finding, population 111, which consisted of cells with low ras p21 and low HER-2ineu expression, included only the diploid cell component and the hyperdiploid cell component to the right of the notch (Fig. 8C). Thus this analysis suggests that paraformaldehyde/methanol fixation resulted in differential propidium iodide staining of a biologically distinct cell subpopulation that would otherwise stain as diploid in samples fixed with alcohol alone.

DISCUSSION The data presented in this study suggest that sequential paraformaldehyde and methanol is a useful fixation procedure for the simultaneous measurement of cellular DNA content using propidium iodide, and cell surface proteins and/or intracellular proteins using immunofluorescent antibody probes. The finding that paraformaldehydelmethanol fixation results in significantly greater tubulin immunofluorescence than methanol alone is consistent with the premise that paraformaldehyde, which crosslinks intracellular proteins, may prevent their leakage of out of cells after cell permeabilization with methanol. The observation that paraformaldehyde followed by methanol results in sig-

441

FIXATIOh' METHOD FOR MULTIPARAMETER FCM ANALYSIS

Table 5 D N A Indices of Diploid and Aneuploid Peaks i n Human Tumor Cells Fixed W i t h Methanol or Paraformaldehydelmethanol, Using a Methanol-Fixed Normal Lymphocyte Reference Standard" DNA Index Mean DNA index of diploid Go/, peaks 0.99 ? 0.02 0.73 _t 0.21

N Methanol Paraformaldehydelmethanol P value (Wilcoxon matched pairs signed rank test)

21

21

N 12 12

Mean DNA index of aneuploid Go,l peaks 1.76 -+ 0.73 1.18 2 0.64

0.0005

0.001

"Tumor cells from patients with various solid tumors and cells obtained from human tumor cell lines cultured in vitro were fixed in 70% methanol or in 0.25% paraformaldehyde and 70% methanol. Following fixation, cells were stained with propidium iodide, 50 pgiml, in PBS containing 1 mglml RNase. To calculate the DNA index, diploid and aneuploid Go,l peak channel numbers were divided by the peak G,,, channel number of methanol-fixed normal diploid lymphocytes. Table 6 D N A Indices of Diploid and Aneuploid Peaks of H u m a n Tumor Cells Fixed W i t h Methanol or Paraformaldehydelmethanol, Using Comparably Fixed Normal Lymphocyte Reference Standards"

Methanol Paraformaldehydelmethanol P value (Wilcoxon matched pairs signed ranks test)

N 25 25

Mean DNA index or diploid GO/, peaks ? S.D. 1.00 k 0.03 1.02 0.10

*

N 16 16

0.13

Mean DNA index i- S.D. of all aneuploid Go,, peaks 1.53 2 0.62 1.77 2 0.82

0.017

N

9 9

Mean DNA index k S.D. of subset of aneuploid Go,, peaks with DI 2 1.5 1.13 2 0.24 1.23 i 0.36

0.374

N 7 7

Mean DNA index 2 S.D. of subset of aneuploid Go,, peaks with DI > 1.5 2.05 2 0.58 2.46 2 0.73

0.03

"Methanol and paraformaldehydeimethanol fixed cells from patients with various tumors were stained with propidium iodide, 50 kglml and 1mg RNaseiml. For each fixation protocol, DNA indices ofthe diploid Goi, peaks were determined as the ratio of the Gall peak channel number and the channel number of the Go,l peak of comparably fixed lymphocytes run separately. The DNA index of the aneuploid Go,, peak in each sample was calculated using the internal diploid reference in that sample.

Table 7 Comparison of Coefficients of Variation o f Diploid Go,,l Peak Measurements in Methanol-Fixed or Paraformaldehydelmethanol -fixed Normal Lymphocytes and i n Clinical Tumors Samples"

Methanol Paraformaldehyde,' methanol P value (Student's t-test. 2-tailed)

Normal lymphocytes Mean CV (%) 2 S.D. 2.53 i 0.94 2.57 2 0.65

Diploid cells in tumor samples Mean CV (96)& S.D. 6.1 2 2.4 7.3 & 3.1

,839

.008

"Cell suspensions from patient tumor samples and tumor cell lines were fixed with 70% methanol or 0.25% paraformaldehyde and 70% methanol, and stained with propidium iodide, 50 kglml, with 1 mglml Rnase.

nificantly greater tubulin immunofluorescence than methanol followed by paraformaldehyde fixation also supports this premise. Morkve and Hostmark (18)have reported similarly that ethanol-fixed tumor cells exhibited reduced p53 protein content in comparison with cells fixed with paraformaldehydeiethanol. It seems likely that the relatively low levels of intra-

cellular immunofluorescence that were observed following fixation with paraformaldehyde alone were due to the failure of the fluoresceinated antibody probe to gain entry into the cells. Our studies suggest that methanol is effective as a cell membrane permeabilization agent, permitting the entry of fluorescent antibody probes into cells. Unlike Triton X-100, methanol effectively permeabilizes cell membranes while also preserving cell light scatter properties. This feature is of considerable importance in immunophenotyping studies of circulating lymphoid and myeloid cells, where gated light scatter measurements are used to identify specific cell subpopulations. Both the concentration of paraformaldehyde and the temperature of paraformaldehyde fixation affect intracellular protein immunofluorescence, cell light scatter properties, and cellular DNA fluorescence measurements using propidium iodide. As noted by Mann e t al. (171, increased intracellular protein immunofluorescence occurs a t high paraformaldehyde concentrations, and a t a fixation temperature of 37°C. These authors have recommended fixation with 2% paraformaldehyde a t 37°C for the measurement of intracellular proteins using immunofluorescent antibody probes. Our findings would suggest that these conditions would, indeed,

442

POLLICE ET AL.

"i

Table 8

S Phase Cell Fraction in Paired Methanol-fixed or Paraformaldehydeimethanol -fixed Samples"

Methanol 7.9 7.9 14.5 14.5 5.4 18.2 18.2 22.6 22.6 5.0 5.0 2.2 2.2

a

S Phase ('"0) Paraformaldehydei methanol 9.9 8.3 14.3 16.0 16.9 9.4 11.1 27.4 28.6 16.4 18.1 7.5 9.5

P

= 0.064 (Wilcoxon Matched-Pairs Signed-Ranks Test)

0

A

DNA \c

0 a,

c

f 10 a,

- +

2

6

4

"Methanol-fixed and paraformaldehydei methanol-fixed cells from cell lines and patients samples were stained with propidium iodide, 50 Kigml, and 1mg RNaseiml. S-phase fractions were calculated using the Modfit Program (Verity Software, Topsham, ME).

be appropriate for single parameter studies. However, these fixation conditions would be suboptimal with regard to preservation of cell light scatter properties and with regard to whole cell DNA studies. For multiparameter studies, paraformaldehyde concentrations of 0.25%and a lower fixation temperatures than 37°C are recommended. Under these conditions, cell light scatter properties are largely preserved, and fixation-dependent variability in DNA dye staining of diploid cells are relatively small. Our studies demonstrate that antibody-impregnated beads can serve as a useful external reference standard for controlling interexperimental variability in instrument performance and day-to-day variations in immunofluorescent probe binding and fluorescence. Adequate control of these variables enabled the detection of reproducible, statistically significant differences in cell immunofluorescence that depend on cell fixation conditions. The regular use of such external reference standards may also permit the flow cytometric detection of quantitative, biologically significant intrastudy differences in the levels of intracellular proteins between tumors and normal tissues, and among different tumors. Our finding that paraformaldehyde fixation can produce spurious aneuploidy in normal leukocytes circulating in peripheral blood has also been noted by others (4,171; this has been attributed to differential DNA staining by monocytes. However, our studies suggest that CD15-positive cells account for only a small proportion of the cells that contribute to these spurious

2

0

100

10'

102

HER-2heu

103

ras

FIG.6. Single parameter histograms in a multiparameter study of tumor cells from a patient with breast cancer. A. DNA histogram of cells fixed in 70% methanol. B. DNA histogram of cells fixed in 0.25% paraformaldehyde and 7 0 8 methanol. C. Histogram of HER-2/neu oncogene protein product per cell in paraformaldehyde/methanolfixed cells. Note log scale for the abscissa. D. Histogram of ras p21 protein per cell in paraformaldehydeimethanol-fixedcells. Note linear abscissa scale. For discussion, see text.

peaks. The adverse effects of paraformaldehyde fixation on DNA measurements (migration of diploid Go,, peak position, increased Gofl coefficient of variation, and spurious aneuploidy) can be minimized by employing low paraformaldehyde concentrations and by avoiding high paraformaldehyde fixation temperatures. We regularly use a paraformaldehyde concentration of 0.25% at fixation temperatures of 25°C or less. Under these fixation conditions there is a relatively small decrease in FITC-labeled antitubulin antibody immunofluorescence (Table Z ) , the effects on propidium iodide staining of cellular DNA are minimized (Tables 3, 41, and cell light scatter properties are well preserved (Fig. 3). We have observed paraformaldehyde concentrationdependent differential DNA staining of aneuploid peaks in human tumors. These differences in DNA staining may be due to variations in dye binding in aneuploid cells, resulting from differing degrees of chromatin condensation and differing degrees of paraformaldehyde-induced nucleoprotein crosslinking and/ or DNA denaturation (2,24).Such fixation proceduredependent effects may also have a bearing on the quality of DNA measurements of cell nuclei obtained

FIXATION METHOD FOR MULTIPARAMETER FCM ANALYSIS

448

_ _ _ _ _ _ _ _ _ _ B-

+ 0

32

16

0

48

32

16

0

48

FIG.7. Dual parameter histograms with gate contours for population subsets of paraformaldehyde/methanol-fixed breast cancer cells. For single parameter histograms, see Figure 6. A. Dual parameter histogram of ras p21 protein per cell vs. cellular DNA plotted as a contour map. Shaded area defines the gated region for population I, a subset of low ras p21, hyperdiploid cells. B. Dual parameter histogram of ras p21 protein per cell vs. cellular DNA. Shaded area defines

32

16

DNA

DNA

48

ras

the gated region for population 11, a subset of increased ras p21, hyperdiploid cells. C. Dual parameter histogram of HER-2ineu oncogene protein per cell vs. ras p21 protein per cell, plotted as a contour map. Shaded area defines the gated region for population 111, a subset of cells with low HER-2heu protein per cell and low ras p21 protein per cell. For discussion, see text.

C

6 v) -

0

0

c o 4 c

P .c

0

w

C

2

a,

2 0

60

120

180

0

60

120

180

0

60

120

180

DNA FIG.8. DNA histograms of populations 1, 11, and 111, as defined by gates shown in Figure 7. A. Population I consists entirely of cells with low ras p21 per cell that occupy the portion of the hyperdiploid peak that lies to the right of the notch in the peak. B. Population I1 consists of cells with increased ras p21 protein per cell most of which occupy the portion ofthe hyperdiploid peak that lies to the left of the notch in the peak. There is some contamination of this subset by overlapping cells from population I. C. Population 111 consists of cells with low

HER-2/neu per cell and low ras p21 per cell. All of these cells are contained in the diploid peak and in the region to the right of the notch in the hyperdiploid peak. The cells to the left of the notch in the hyperdiploid peak are excluded from population 111, confirming that the hyperdiploid peak consists of two biologically distinct populations with slightly different, but overlapping propidium iodide-staining characteristics. For further discussion, see text.

from formalin-fixed, paraffin-embedded tissues. In order to minimize any difficulties in estimating DNA indices of aneuploid cells that might result from paraformaldehyde-induced differential changes in propidium iodide DNA staining, we recommend the use of DNA histograms obtained from alcohol-fixed cells as the reference standards for quantitating and reporting DNA indices in aneuploid DNA histograms. Paraformaldehyde-induced differential propidium iodide staining of abnormal cells may actually prove to be useful a s a technique for identifying biologically distinctive populations that might otherwise be indistinguishable from diploid cells in ethanol-fixed samples. An example of the identification of such a population in a paraformaldehydeimethanol-fixed sample is given in Figures 6-8. Clearly, additional studies comparing alcohol-fixed cells and paraformaldehyde-fixed cells from the same sample must be done to further evaluate the

potential utility of paraformaldehyde-induced differential propidium iodide DNA staining in flow cytometric studies of tumors.

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