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An easy and economical method to prepare cells for cytologic analyses
ELSEVIER
An Easy and Economical Method to Prepare Cells for Cytologic Analyses Brian J. Day, Abram Division
of Critical
Hatch,
Care and Respiratory Medicine,
Donna
Department
D. Carstens, of Medicine,
and James D. Crapo
Duke University, Durham,
North Carolina,
U.S.A.
A crucial step in any cytology application begins with the placement of cells on slides in a manner that provides good preservation of cell morphology. Commercial cytocentrifuges are the most commonly used devices for this purpose. However, there are instances when these devices are either not available or not applicable. We devised an easy and economical alternative method to prepare cells for cytologic analysis. The device (cytograv) was easily assembled from common laboratory and office supplies, and produced cell preparations of similar quality as those produced with a cytocentrifuge (cytospin-3). Cellular analyses of biological fluids is one of the most common applications for these kinds of devices. The cytograv device was successfully employed in the identification and verification of a PMN isolation procedure from whole blood. The cytograv device was also successfully used to quantitate increases in the number of PMNs in bronchoalveolar lavage fluid recovered from mice treated with increasing doses of paraquat. These two examples illustrate some of the many possible uses of the cytograv device to provide high-quality preparations for cytologic analyses. Key Words:
Paraquat; Cell isolation; Cytology; PMN; Bronchoalveolar lavage
Introduction Cytology of biological fluids plays an important role in the diagnosis and monitoring of infectious diseases and in the diagnosis of many neoplasms. Changes in the
number and types of white blood cells are frequently used as markers of infection and/or inflammation. Toxicologists have applied this concept to study the toxic effects of xenobiotics on biological systems. One example involves the use of bronchoalveolar lavage to sample lung epithelial lining fluids for alterations, produced by xenobiotics, in leukocyte numbers and types as markers of inflammation and immune responses (National Research Council, 1989). Bronchoalveolar lavage fluid (BALF) analysis is commonly used to semiquantitate lung injury. The most commonly used BALF markers of injury include increases in lactate dehydrogenase (LDH) to assesscell injury and increases in protein levels, which indicate edema. Many airborne toxicants have been found to alter the number
and types of leukocytes in
Address reprint requests to Brian J. Day, Ph.D., Box 3177, Division of Critical Care and Respiratory Medicine, Duke University Medical Center, Durham, NC 27710, U.S.A. Received January 24, 1995; revised and accepted March 22, 1995. Journal of Pharmacological and Toxicological Methods 0 1995 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
34, 57-62
bronchoalveolar lavage fluids, including cytotoxic agents like paraquat (Schoenberger et al., 1984; Hampson et al., 1989) and ozone (Seltzer et al., 1986; Koren et al., 1989) as well as substances known to produce altered immune responses, such as beryllium (Epstein et al., 1982) and isocyanates (Karol, 1986; Gupta et al., 1991). Preparations of cytological specimens from biological fluids are required to identify specific cell types and/or perform differential cell counts. Many techniques have been proposed for the preparation of cytological specimens for analyses (Mayhew and Beal, 1980). However, most of these require complicated or expensive equipment. The most common technique employs centrifugation to allow for rapid preparation of samples with good cytomorphological preservation. These devices use centrifugal force to concentrate cells onto slides. Alternative methods employ sedimentation of cells onto slides by gravitational force. We now describe a simple cytograv system that is a sedimentation-based
device that can be
quickly assembled with common laboratory and office supplies, and produce preparations similar in quality to those prepared by more costly, commercially available cytocentrifuges. The cytograv system provides a unique, simplistic alternative to other sedimentation systems and
(1995) SSDI
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58
JPM Vol. 34, No. 1 September 199557-62
expensive cytocentrifuges for preparing cytological specimens. This study describes the proper assembly and use of the cytograv device in the isolation of polymorphonuclear (PMN) leukocytes from whole blood and in BALF analysis of paraquat-induced lung injury.
Materials
and Methods
Materials Materials needed for assembly of the cytograv device includes #l filter papers 4.25 cm (Whatman, Maidstone, England), medium binder clips (Office Mate, Edison, NJ), glass slides, Tygon tubing (Fischer Scientific, Pittsburgh, PA), l-cc syringes (Becton Dickinson, Franklin Lakes, NJ), and a paperpunch (McGill, Marengo, IL). Chemicals used include phosphate buffered saline, trypan blue (Gibco, Grand Island, NY), leukostat stain, (Fisher Scientific, Pittsburgh, PA), coomassie plus protein reagent (Pierce, Rockford, IL), pentobarbital (Abbot, Chicago, IL), pyruvate, NADH, histopaque density gradients, and paraquat (Sigma, St. Louis, MO). Animals Twenty Swiss-Webster male mice (21-28 g), six C57BW6J male mice (25-30 g), and two SpragueDawley male rats (300-350 g) were used in these studies. Animals were housed in the Duke Vivarium. Animals were given chow and tap water ad libitum and acclimated to 22” C in environmentally controlled rooms (12-hr light cycles) at least 6 days prior to treatment. Animal use was reviewed and approved by the Duke Animal Use Committee, which follows recommended guidelines reported in the Guide for the Care and Use of LaboratoT Animals (DHEW, 1978). Animal Study Designs In the paraquat toxicity study, groups containing five mice were treated with 0, 35, 45, or 55 mg/kg body weight, i.p., of paraquat in saline (10 mL/kg). Measurements were made 48 hr after the administration of paraquat. Mice that died before 48 hr were excluded from the study. Bronchoalveolar Lavage Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the trachea was cannulated. A midline incision was made, and the diaphragm was punctured. Mice were lavaged twice with 1 mL of calcium and magnesium-free PBS. Recovered bronchoalveolar lavage fluid (BALF) was pooled and used for biochemical assays and cytology.
PMN Isolation Rats were anesthetized with pentobarbital(50 mg/kg, i.p.), and a midline incision was made to expose the diaphragm. The diaphragm was punctured, and 8 cc of blood was drawn via a cardiac puncture into EDTA containing tubes. Neutrophils were isolated using a modified procedure described by English and Anderson (1974). Briefly, blood was centrifuged at 2208. The plasma and buffy coat was removed and diluted with an equal volume of PBS without calcium or magnesium and layered over two histopaque gradients (1.077 and 1.119, respectively). Tubes were centrifuged at 8OOgfor 20 min at 25” C. Neutrophils were collected from the second interface layer. Biochemical Assays BALF was centrifuged at 8OOg for 15 min at 25” C, and the cell-free supernatant was analyzed for lactate dehydrogenase (LDH) activity, using a method reported by Vassault (1983) that was adapted for a plate-reader format (Thermomax Plate Reader, Molecular Devices, Sunnyvale, CA), and total proteins using the Coomassie Plus assay (Pierce, Rockford, IL). Cytology The cell pellet from the BALF was resuspended in PBS and an aliquot counted with a hemacytometer. Approximately 5 X lo4 cells were loaded onto the cytograv device in 125~FL volume. After approximately 15 to 20 min the cells are fixed to the slides and are ready for staining. A similar volume and number of cells were loaded into the cytofunnel of a cytospin-3 (Shandon, Pittsburgh, PA) using SCA-0005 filter cards and spun at 1000 rpm for 3 min. Cells were stained with a modified Wright’s stain (Leukostat, Fisher Scientific, Pittsburgh, PA) for differential cell counting. In the paraquat study, three fields of 100 cells were counted as the number of PMNs/lOO nucleated cells and expressed as a percentage. Data Analysis All experiments were analyzed by one-way analysis of variance (ANOVA) and Tukey’s multiple range test using a computer statistical program (InStat, GraphPad Software, San Diego, CA). The acceptable level of significance was established at p < .05.
Results Assembly and Use of the Cytograv Device The cytograv device is easily assembled from common office and laboratory supplies, as shown in Figure 1A.
B.J. DAY METHOD
ET AL. FOR CYTOLOGIC
59 ANALYSES
cell density on the slide. This can be determined by counting the number of cells in a suspension and serially diluting them before placing them on the slide with the cytograv device. The cytospin is a commercially available device that uses centrifugal force to place cells on slides in a small area. A comparison was made between slides prepared with a cytospin and slides prepared with the cytograv device from bronchoalveolar lavage cells obtained from mice (Figure 2). Both devices produced similar results with respect to cell morphology and density.
Use of Cytogravin the Isolation of Blood Leukocytes There are many situations in which one needs to determine what types of cells are in a biological fluid or if an isolation of one type of cell from others is success1. Assembly of the cytograv device. (A) Materials needed for the assembly of the cytograv device. (B) An assembled cytograv device that is ready for use.
Figure
The first step involves making a center hole in a filter paper with a hole punch. Next, a l-cc syringe barrel is cut in half with a razor blade. Save the top half (flanged portion) of the syringe barrel. The filter paper is centered on a clean glass slide, and the barrel of the syringe is then carefully aligned, the flanges perpendicular to the slide, with the center hole in the filter paper and clamped into place with two binder clips. The clips’ edges are fitted with Tygon tubing (6-mm o.d./3-mm i.d.) with a notch in the center of each top edge. The flanged portion of the syringe barrel fits in the notched portion of the tubing. The tubing prevents the clips from scratching or fracturing the glass slide. The cytograv device is now ready for use and should look like the one pictured in Figure 1B.
To use the device, place it on a flat surface and prime it by adding about 20 FL of the same fluid in which the cells are suspended into the syringe barrel. Next, add up to 150 ~J,L of cell suspension to the syringe barrel. If larger volumes are required, then increase the number of filter papers per slide. Wait until all the fluid has been absorbed before dismantling the device. This should take 15 to 20 min to occur. If this occurs faster than 10 min, it may be due to improper alignment between the filter paper hole and syringe barrel, and may cause uneven distribution of cells on the slide. Be especially careful not to disturb the adherent cell center. The cell center should be completely dry before any cell staining
is done, to prevent cell loss during the staining procedure. Depending on the use, one may need more or less
2. Comparison of cytospin versus cytograv. Mouse BALF cytology from: (A) cytograv preparation; (B) cytospin preparation. Notice that the morphology of the cells are very comparable between preparations. Magnification bars = 50 micrometer km.
Figure
60
ful. An example of the latter is the isolation of polymorphonuclear (PMN) leukocytes from other blood cells. A common way to isolate PMNs from other leukocytes involves the use of density gradients. Figure 3 shows an enriched population of PMNs separated from rat blood using histopaque density gradients. Cells from the second interface were placed on slides using the cytograv device and stained with a modified Wright’s stain. PMNs are commonly separated and used in vitro as generators of oxidative stress in cell culture inflammation models. This is an example where the cytograv device allows one to verify the purity of separated cell samples with minimum consumption of cells, allowing the majority of the sample to be used for in vitro studies.
Use of Cytograv in BALF Analysis A common use of cytology involves the assessment of biological fluids for markers of pathological changes in white blood cell number and types. PMNs in the BALF are normally a rare event; however, after inflammatory episodes their numbers increase dramatically. To illustrate this, mice were treated with a known pneumotoxicant, paraquat, and BALF from these mice were analyzed for a variety of common biomarkers of lung injury along with changes in the number of PMNs. Bronchoalveolar cells were loaded on the cytograv and stained, and differential cell counts were performed. Figure 4 depicts representative micrographs from each of the treatment groups showing a relative increase in the number of PMNs per field with respect to paraquat dose. Paraquatinduced lung injury produced a dose-dependent increase in percentage of PMN, LDH, and protein (Table 1). This example shows the utility of using cytology to assesslung
Figure 3. Polymorphonuclear (PMN) cell cytology using a cytograv device. Rat PMNs were separated from other leukocytes using h&opaque density gradients. PMNs (arrowheads) were stained using a modified Wright’s stain. Magnification bar = 50 micrometer km.
JPM Vol. 34, No. September 199557-62
injury by helping define inflammatory pathophysiology.
1
components in the
Discussion Most cytological samples are presently prepared using commercial cytocentrifuges that produce samples with good cytomorphological preservation. However, there are instances when these devices are either not available or not applicable, and sedimentation-based devices have been used as an alternative. A variety of sedimentation devices have been described in the literature (Kolar and Zeman, 1968; Meyhew and Beal, 1980; Cook and DeNicola, 1988). However, many of these systems are awkward or complicated in design, assembly, and operation. The cytograv device was modeled from a sedimentation device previously described by Cook and DeNicola (1988). Their device used a wooden base mounted with four bolts and two strips of wood to clamp a syringe barrel onto the filter paper and slide with wing nuts. Considerable time and skill was required to properly assemble the device. The wing nuts had to be tightened and loosened carefully to avoid breaking the slide. The cytograv is a much more efficient version that does not require custom parts and can be rapidly assembled and disassembled. Cytograv is a good alternative for small laboratories that cannot afford expensive cytocentrifuges, or it can be used as a backup. The only disadvantage cytograv has over cytocentrifuges is it takes longer for sample preparation. However, in many cases the cytograv device has several advantages over commercial cytocentrifuges. The cytograv possesses no moving parts, which prevents breakdowns, and has no set limits on the number of slides handled per run. The cytograv is disposable because all its parts are inexpensive. This may prove advantageous to investigators who use fluids that are radioactive or biohazardous. Other advantages the cytograv possesses are its simplicity and its portability. This may be advantageous to investigators who do fieldwork where a laboratory is not accessible. Preparation of cytological samples is often required during the isolation of one cell type from a population of different cell types. These samples are used to identify cells of interest and verify the isolation techniques. A cytograv preparation has the advantage over a smear preparation in that it takes fewer cells because they are concentrated in a small area of the slide. The applications for cytograv are virtually endless, including the identification and verification of cells separations (English and Anderson, 1974) as well as cells isolated from tissue digests (Rannels and Rannels, 1988) and biological fluids (Reynolds, 1987). The example used in this paper involved the separation of PMNs from whole blood. Activated PMNs are commonly used in cell
61
B.J. DAY ET AL. METHOD FOR CYTOLOGIC ANALYSES
Figure 4. Bronchoalveolar lavage fluid (BALF) cytology from paraquat-treated mice. Representative BALF cytology obtained from mice given increasing amounts of paraquat (i.p.) and killed 48 hr later: (A) control; (B) 35 m&g; (C) 45 mg/kg; (D) 55 mg/kg. Note the increase in numbers of PMNs in the higher paraquat doses. Magnification bar = 50 micrometer km.
Table 1. Effect of Paraquat on Bronchoalveolar Lavage Fluid Biomarkers” Paraquat (mgikg, i.p.) 0 35 45 55
N 5 5 5 3f
%PMN 1.3 k 1.8 k 8.3 k 8.7 2
0.6b l.lb 1.7’ 1.8’
LDH (U/L) 31.5 59.8 102.5 214.6
2 ? k k
4.0b 8.9’ 7.6d 59.7’
Protein @giW 10.1 2 O.gb 17.1 2 3.8’ 54.5 2 5.7d 62.2 2 8.3*
“Mice were treated with various doses of paraquat and then killed 48 hr later. Results are expressed as the mean 2 SE. b-eValues with different superscripts are significantly different from one another, p < .05, as determined using a one-way ANOVA with a Tukey’s multiple comparisons test. fTwo mice died in this treatment group before 48 hr and were excluded from the study.
culture models of inflammation (Weiss et al., 1981; Simon et al., 1986). The cytograv device provided cytological preparations with good morphological preservation that allowed easy verification of the purity of cell
separation with minimal loss of cells needed for other purposes. Bronchoalveolar lavage fluid (BALF) analysis has gained popularity as a rapid in vivo screen to evaluate the toxicity of both systemic and inhaled pneumotoxicants (Henderson et al., 1985). Many pneumotoxicants produce inflammatory reactions in the lung that can be better assessed by measuring changes in BALF leukocytes (National Research Council, 1989; Day et al., 1990). Paraquat is thought to produce lung injury, in part, through its ability to generate reactive oxygen species by redox cycling with cellular NADPH diaphorases and oxygen (Bus et al., 1974). Paraquat poisoning produces a well-characterized injury to alveolar epithelium and endothelium along with an inflammatory response consisting of PMN and fibroblast infiltration (Brooks, 1971). In this study, paraquat produced an increase in the number of lavagable PMNs that was consistent with paraquat’s known pathophysiologic response in the lung. Paraquat was employed to illustrate
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how BALF analysis, with the aid of cytograv, can allow one to quantitate a pneumotoxicant’s ability to generate an inflammatory response. In summary, the cytograv method provided an easy and economical alternative to commercial cytocentrifuges. The cytograv method has many advantages over both cytocentrifuges and other sedimentation devices previously reported. These combined features of the cytograv should provide a valuable tool for investigators who deal with cytology of biological fluids, cell isolation, and identifications. The authors thank Dr. Vuokko Kinnula, Neilson for their advice and able technical supported, in part, by PO1 HL 31992.
Kathy Evans, assistance. This
and Bob study was
of lung lesions produced by ingested paraquat on mouse lung. Lab Invest
Bus JS, Aust SD, Gibson JE (1974) Superoxide and singlet oxygencatalyzed lipid peroxidation as a possible mechanism for paraquat (methyl viologen) toxicity. Biochem Siophys Res Commun 58:749754. Cook
JR, DeNicola 18:475-499.
DB (1988)
Cerebrospinal
fluid.
Vet Clin North
Am
Day BJ, Carlson GP, DeNicola DB (1990) gamma-Glutamyltranspeptidase in rat bronchoalveolar lavage fluid as a probe of 4-ipomeanol and alpha-naphthylthiourea-induced pneumotoxicity. J Pharmacol Methods 24:1-8. DHEW (1978) In Guide for the Care and Use of Laboratoy National Institute of Health, Publication No. 78-23. English D, Anderson cells: Granulocytes density gradient
Animals.
BR (1974) Single-step separation of red blood and mononuclear leukocytes on discontinuous of ficoll-hypaque. J Immunol Methods 51249-257.
Pond SM (1989) Effects of paraquat lavage fluid. Toxicol Appl Pharmacol
on 98:
Henderson RF, Benson JM, Hahn FF, Hobbs CH, Jones RK, Mauderly JL, McClellan RO, Pickrell JA (1985) New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis. Fundam Appl Toxic01 5:451-458. Karol MH (1986) Respiratory Rev Toxic01 16:349-379. Kolar
0, Zeman 18:44-51.
V (1968)
effects Spinal
of inhaled
fluid
isocyanates.
cytomorphology.
CRC Arch
Crit
Neural
Koren HS, Devlin RB, Graham DE, Mann R, McGee MP, Horstman DH, Kozumbo WJ, Becker S, House DE, McDonnell WF, Bromberg PA (1989) Ozone-induced inflammation in lower airways of human subjects. Am Rev Respir Dis 139:407-415. Mayhew fluid.
References Brooks RE (1971) Ultrastructure chemicals: Effect of herbicide 251536545.
Hampson EC, Eyles DW, canine bronchoalveolar 206-215.
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IG, Beal CR (1980) Techniques Vet Clin North Am 10:155-159.
of analysis
of cerebrospinal
National Research Council (1989) Markers of inflammatory and immune response. In Biologic Markers in Pulmonary Toxicology. Washington, DC: National Academy Press, pp. 91-103. Rannels SR, Rannels DE (1988) Isolation and culture of alveolar type II cells for toxicological studies. In Toxicology of the Lung. Ed., DE Gardner, JD Crapo, and EJ Massaro. New York: Raven Press, pp. 219-238. Reynolds HY 135:250-263.
(1987)
Bronchoalveolar
lavage.
Am
Rev
Respir
Dis
Schoenberger CI, Rennard SI, Bitterman PB, Fukuda Y, Ferrans VJ, Crystal RG (1984) Paraquat-induced pulmonary fibrosis: Role of the alveolitis in modulating the development of fibrosis. Am Rev Respir Dis 129:168-173. Seltzer J, Bigby BG, Stulbarg M, Holtzman MJ, Nadel JA, Ueki IF, Leikauf GD, Goetzl ET, Boushey HA (1986) Ozone-induced change in bronchial reactivity to methacholine and airway inflammation in humans. .7 Appl Physiol 60:1321-1326. Simon RH, DeHart PD, Todd RF (1986) Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. J Clin Invest 78:1375-1386.
Epstein PE, Dauber JH, Rossman MD, Daniele RP (1982) Bronchoalveolar lavage in a patient with chronic berylliosis: Evidence for hypersensitivity pneumonitis. Ann Intern Med 97:213-216.
Vassault A (1983) Lactate dehydrogenase. Analysis. Ed., HU Bergmeyer. New 118-126.
In Methods of Enzymatic York: Academic Press, pp.
Gupta GS, Kaw JL, Naqvi SH, Dixit R, Ray PK (1991) Inhalation toxicity of methyl isocyanate: Biochemical and cytological profile of bronchoalveolar lavage fluid in rats. JApp/ Toxic01 11:157-160.
Weiss SJ, Young J, LoBuglio AF, Slivka A, Nimeh NF (1981) Role of peroxide in neutrophil-mediated destruction of cultured endotheha1 cells. J Clin Invest 68:714-721.
An Easy and Economical Method to Prepare Cells for Cytologic Analyses Brian J. Day, Abram Division
of Critical
Hatch,
Care and Respiratory Medicine,
Donna
Department
D. Carstens, of Medicine,
and James D. Crapo
Duke University, Durham,
North Carolina,
U.S.A.
A crucial step in any cytology application begins with the placement of cells on slides in a manner that provides good preservation of cell morphology. Commercial cytocentrifuges are the most commonly used devices for this purpose. However, there are instances when these devices are either not available or not applicable. We devised an easy and economical alternative method to prepare cells for cytologic analysis. The device (cytograv) was easily assembled from common laboratory and office supplies, and produced cell preparations of similar quality as those produced with a cytocentrifuge (cytospin-3). Cellular analyses of biological fluids is one of the most common applications for these kinds of devices. The cytograv device was successfully employed in the identification and verification of a PMN isolation procedure from whole blood. The cytograv device was also successfully used to quantitate increases in the number of PMNs in bronchoalveolar lavage fluid recovered from mice treated with increasing doses of paraquat. These two examples illustrate some of the many possible uses of the cytograv device to provide high-quality preparations for cytologic analyses. Key Words:
Paraquat; Cell isolation; Cytology; PMN; Bronchoalveolar lavage
Introduction Cytology of biological fluids plays an important role in the diagnosis and monitoring of infectious diseases and in the diagnosis of many neoplasms. Changes in the
number and types of white blood cells are frequently used as markers of infection and/or inflammation. Toxicologists have applied this concept to study the toxic effects of xenobiotics on biological systems. One example involves the use of bronchoalveolar lavage to sample lung epithelial lining fluids for alterations, produced by xenobiotics, in leukocyte numbers and types as markers of inflammation and immune responses (National Research Council, 1989). Bronchoalveolar lavage fluid (BALF) analysis is commonly used to semiquantitate lung injury. The most commonly used BALF markers of injury include increases in lactate dehydrogenase (LDH) to assesscell injury and increases in protein levels, which indicate edema. Many airborne toxicants have been found to alter the number
and types of leukocytes in
Address reprint requests to Brian J. Day, Ph.D., Box 3177, Division of Critical Care and Respiratory Medicine, Duke University Medical Center, Durham, NC 27710, U.S.A. Received January 24, 1995; revised and accepted March 22, 1995. Journal of Pharmacological and Toxicological Methods 0 1995 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
34, 57-62
bronchoalveolar lavage fluids, including cytotoxic agents like paraquat (Schoenberger et al., 1984; Hampson et al., 1989) and ozone (Seltzer et al., 1986; Koren et al., 1989) as well as substances known to produce altered immune responses, such as beryllium (Epstein et al., 1982) and isocyanates (Karol, 1986; Gupta et al., 1991). Preparations of cytological specimens from biological fluids are required to identify specific cell types and/or perform differential cell counts. Many techniques have been proposed for the preparation of cytological specimens for analyses (Mayhew and Beal, 1980). However, most of these require complicated or expensive equipment. The most common technique employs centrifugation to allow for rapid preparation of samples with good cytomorphological preservation. These devices use centrifugal force to concentrate cells onto slides. Alternative methods employ sedimentation of cells onto slides by gravitational force. We now describe a simple cytograv system that is a sedimentation-based
device that can be
quickly assembled with common laboratory and office supplies, and produce preparations similar in quality to those prepared by more costly, commercially available cytocentrifuges. The cytograv system provides a unique, simplistic alternative to other sedimentation systems and
(1995) SSDI
1056.87191951$9.50 1056-8719(95)00029-H
58
JPM Vol. 34, No. 1 September 199557-62
expensive cytocentrifuges for preparing cytological specimens. This study describes the proper assembly and use of the cytograv device in the isolation of polymorphonuclear (PMN) leukocytes from whole blood and in BALF analysis of paraquat-induced lung injury.
Materials
and Methods
Materials Materials needed for assembly of the cytograv device includes #l filter papers 4.25 cm (Whatman, Maidstone, England), medium binder clips (Office Mate, Edison, NJ), glass slides, Tygon tubing (Fischer Scientific, Pittsburgh, PA), l-cc syringes (Becton Dickinson, Franklin Lakes, NJ), and a paperpunch (McGill, Marengo, IL). Chemicals used include phosphate buffered saline, trypan blue (Gibco, Grand Island, NY), leukostat stain, (Fisher Scientific, Pittsburgh, PA), coomassie plus protein reagent (Pierce, Rockford, IL), pentobarbital (Abbot, Chicago, IL), pyruvate, NADH, histopaque density gradients, and paraquat (Sigma, St. Louis, MO). Animals Twenty Swiss-Webster male mice (21-28 g), six C57BW6J male mice (25-30 g), and two SpragueDawley male rats (300-350 g) were used in these studies. Animals were housed in the Duke Vivarium. Animals were given chow and tap water ad libitum and acclimated to 22” C in environmentally controlled rooms (12-hr light cycles) at least 6 days prior to treatment. Animal use was reviewed and approved by the Duke Animal Use Committee, which follows recommended guidelines reported in the Guide for the Care and Use of LaboratoT Animals (DHEW, 1978). Animal Study Designs In the paraquat toxicity study, groups containing five mice were treated with 0, 35, 45, or 55 mg/kg body weight, i.p., of paraquat in saline (10 mL/kg). Measurements were made 48 hr after the administration of paraquat. Mice that died before 48 hr were excluded from the study. Bronchoalveolar Lavage Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the trachea was cannulated. A midline incision was made, and the diaphragm was punctured. Mice were lavaged twice with 1 mL of calcium and magnesium-free PBS. Recovered bronchoalveolar lavage fluid (BALF) was pooled and used for biochemical assays and cytology.
PMN Isolation Rats were anesthetized with pentobarbital(50 mg/kg, i.p.), and a midline incision was made to expose the diaphragm. The diaphragm was punctured, and 8 cc of blood was drawn via a cardiac puncture into EDTA containing tubes. Neutrophils were isolated using a modified procedure described by English and Anderson (1974). Briefly, blood was centrifuged at 2208. The plasma and buffy coat was removed and diluted with an equal volume of PBS without calcium or magnesium and layered over two histopaque gradients (1.077 and 1.119, respectively). Tubes were centrifuged at 8OOgfor 20 min at 25” C. Neutrophils were collected from the second interface layer. Biochemical Assays BALF was centrifuged at 8OOg for 15 min at 25” C, and the cell-free supernatant was analyzed for lactate dehydrogenase (LDH) activity, using a method reported by Vassault (1983) that was adapted for a plate-reader format (Thermomax Plate Reader, Molecular Devices, Sunnyvale, CA), and total proteins using the Coomassie Plus assay (Pierce, Rockford, IL). Cytology The cell pellet from the BALF was resuspended in PBS and an aliquot counted with a hemacytometer. Approximately 5 X lo4 cells were loaded onto the cytograv device in 125~FL volume. After approximately 15 to 20 min the cells are fixed to the slides and are ready for staining. A similar volume and number of cells were loaded into the cytofunnel of a cytospin-3 (Shandon, Pittsburgh, PA) using SCA-0005 filter cards and spun at 1000 rpm for 3 min. Cells were stained with a modified Wright’s stain (Leukostat, Fisher Scientific, Pittsburgh, PA) for differential cell counting. In the paraquat study, three fields of 100 cells were counted as the number of PMNs/lOO nucleated cells and expressed as a percentage. Data Analysis All experiments were analyzed by one-way analysis of variance (ANOVA) and Tukey’s multiple range test using a computer statistical program (InStat, GraphPad Software, San Diego, CA). The acceptable level of significance was established at p < .05.
Results Assembly and Use of the Cytograv Device The cytograv device is easily assembled from common office and laboratory supplies, as shown in Figure 1A.
B.J. DAY METHOD
ET AL. FOR CYTOLOGIC
59 ANALYSES
cell density on the slide. This can be determined by counting the number of cells in a suspension and serially diluting them before placing them on the slide with the cytograv device. The cytospin is a commercially available device that uses centrifugal force to place cells on slides in a small area. A comparison was made between slides prepared with a cytospin and slides prepared with the cytograv device from bronchoalveolar lavage cells obtained from mice (Figure 2). Both devices produced similar results with respect to cell morphology and density.
Use of Cytogravin the Isolation of Blood Leukocytes There are many situations in which one needs to determine what types of cells are in a biological fluid or if an isolation of one type of cell from others is success1. Assembly of the cytograv device. (A) Materials needed for the assembly of the cytograv device. (B) An assembled cytograv device that is ready for use.
Figure
The first step involves making a center hole in a filter paper with a hole punch. Next, a l-cc syringe barrel is cut in half with a razor blade. Save the top half (flanged portion) of the syringe barrel. The filter paper is centered on a clean glass slide, and the barrel of the syringe is then carefully aligned, the flanges perpendicular to the slide, with the center hole in the filter paper and clamped into place with two binder clips. The clips’ edges are fitted with Tygon tubing (6-mm o.d./3-mm i.d.) with a notch in the center of each top edge. The flanged portion of the syringe barrel fits in the notched portion of the tubing. The tubing prevents the clips from scratching or fracturing the glass slide. The cytograv device is now ready for use and should look like the one pictured in Figure 1B.
To use the device, place it on a flat surface and prime it by adding about 20 FL of the same fluid in which the cells are suspended into the syringe barrel. Next, add up to 150 ~J,L of cell suspension to the syringe barrel. If larger volumes are required, then increase the number of filter papers per slide. Wait until all the fluid has been absorbed before dismantling the device. This should take 15 to 20 min to occur. If this occurs faster than 10 min, it may be due to improper alignment between the filter paper hole and syringe barrel, and may cause uneven distribution of cells on the slide. Be especially careful not to disturb the adherent cell center. The cell center should be completely dry before any cell staining
is done, to prevent cell loss during the staining procedure. Depending on the use, one may need more or less
2. Comparison of cytospin versus cytograv. Mouse BALF cytology from: (A) cytograv preparation; (B) cytospin preparation. Notice that the morphology of the cells are very comparable between preparations. Magnification bars = 50 micrometer km.
Figure
60
ful. An example of the latter is the isolation of polymorphonuclear (PMN) leukocytes from other blood cells. A common way to isolate PMNs from other leukocytes involves the use of density gradients. Figure 3 shows an enriched population of PMNs separated from rat blood using histopaque density gradients. Cells from the second interface were placed on slides using the cytograv device and stained with a modified Wright’s stain. PMNs are commonly separated and used in vitro as generators of oxidative stress in cell culture inflammation models. This is an example where the cytograv device allows one to verify the purity of separated cell samples with minimum consumption of cells, allowing the majority of the sample to be used for in vitro studies.
Use of Cytograv in BALF Analysis A common use of cytology involves the assessment of biological fluids for markers of pathological changes in white blood cell number and types. PMNs in the BALF are normally a rare event; however, after inflammatory episodes their numbers increase dramatically. To illustrate this, mice were treated with a known pneumotoxicant, paraquat, and BALF from these mice were analyzed for a variety of common biomarkers of lung injury along with changes in the number of PMNs. Bronchoalveolar cells were loaded on the cytograv and stained, and differential cell counts were performed. Figure 4 depicts representative micrographs from each of the treatment groups showing a relative increase in the number of PMNs per field with respect to paraquat dose. Paraquatinduced lung injury produced a dose-dependent increase in percentage of PMN, LDH, and protein (Table 1). This example shows the utility of using cytology to assesslung
Figure 3. Polymorphonuclear (PMN) cell cytology using a cytograv device. Rat PMNs were separated from other leukocytes using h&opaque density gradients. PMNs (arrowheads) were stained using a modified Wright’s stain. Magnification bar = 50 micrometer km.
JPM Vol. 34, No. September 199557-62
injury by helping define inflammatory pathophysiology.
1
components in the
Discussion Most cytological samples are presently prepared using commercial cytocentrifuges that produce samples with good cytomorphological preservation. However, there are instances when these devices are either not available or not applicable, and sedimentation-based devices have been used as an alternative. A variety of sedimentation devices have been described in the literature (Kolar and Zeman, 1968; Meyhew and Beal, 1980; Cook and DeNicola, 1988). However, many of these systems are awkward or complicated in design, assembly, and operation. The cytograv device was modeled from a sedimentation device previously described by Cook and DeNicola (1988). Their device used a wooden base mounted with four bolts and two strips of wood to clamp a syringe barrel onto the filter paper and slide with wing nuts. Considerable time and skill was required to properly assemble the device. The wing nuts had to be tightened and loosened carefully to avoid breaking the slide. The cytograv is a much more efficient version that does not require custom parts and can be rapidly assembled and disassembled. Cytograv is a good alternative for small laboratories that cannot afford expensive cytocentrifuges, or it can be used as a backup. The only disadvantage cytograv has over cytocentrifuges is it takes longer for sample preparation. However, in many cases the cytograv device has several advantages over commercial cytocentrifuges. The cytograv possesses no moving parts, which prevents breakdowns, and has no set limits on the number of slides handled per run. The cytograv is disposable because all its parts are inexpensive. This may prove advantageous to investigators who use fluids that are radioactive or biohazardous. Other advantages the cytograv possesses are its simplicity and its portability. This may be advantageous to investigators who do fieldwork where a laboratory is not accessible. Preparation of cytological samples is often required during the isolation of one cell type from a population of different cell types. These samples are used to identify cells of interest and verify the isolation techniques. A cytograv preparation has the advantage over a smear preparation in that it takes fewer cells because they are concentrated in a small area of the slide. The applications for cytograv are virtually endless, including the identification and verification of cells separations (English and Anderson, 1974) as well as cells isolated from tissue digests (Rannels and Rannels, 1988) and biological fluids (Reynolds, 1987). The example used in this paper involved the separation of PMNs from whole blood. Activated PMNs are commonly used in cell
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B.J. DAY ET AL. METHOD FOR CYTOLOGIC ANALYSES
Figure 4. Bronchoalveolar lavage fluid (BALF) cytology from paraquat-treated mice. Representative BALF cytology obtained from mice given increasing amounts of paraquat (i.p.) and killed 48 hr later: (A) control; (B) 35 m&g; (C) 45 mg/kg; (D) 55 mg/kg. Note the increase in numbers of PMNs in the higher paraquat doses. Magnification bar = 50 micrometer km.
Table 1. Effect of Paraquat on Bronchoalveolar Lavage Fluid Biomarkers” Paraquat (mgikg, i.p.) 0 35 45 55
N 5 5 5 3f
%PMN 1.3 k 1.8 k 8.3 k 8.7 2
0.6b l.lb 1.7’ 1.8’
LDH (U/L) 31.5 59.8 102.5 214.6
2 ? k k
4.0b 8.9’ 7.6d 59.7’
Protein @giW 10.1 2 O.gb 17.1 2 3.8’ 54.5 2 5.7d 62.2 2 8.3*
“Mice were treated with various doses of paraquat and then killed 48 hr later. Results are expressed as the mean 2 SE. b-eValues with different superscripts are significantly different from one another, p < .05, as determined using a one-way ANOVA with a Tukey’s multiple comparisons test. fTwo mice died in this treatment group before 48 hr and were excluded from the study.
culture models of inflammation (Weiss et al., 1981; Simon et al., 1986). The cytograv device provided cytological preparations with good morphological preservation that allowed easy verification of the purity of cell
separation with minimal loss of cells needed for other purposes. Bronchoalveolar lavage fluid (BALF) analysis has gained popularity as a rapid in vivo screen to evaluate the toxicity of both systemic and inhaled pneumotoxicants (Henderson et al., 1985). Many pneumotoxicants produce inflammatory reactions in the lung that can be better assessed by measuring changes in BALF leukocytes (National Research Council, 1989; Day et al., 1990). Paraquat is thought to produce lung injury, in part, through its ability to generate reactive oxygen species by redox cycling with cellular NADPH diaphorases and oxygen (Bus et al., 1974). Paraquat poisoning produces a well-characterized injury to alveolar epithelium and endothelium along with an inflammatory response consisting of PMN and fibroblast infiltration (Brooks, 1971). In this study, paraquat produced an increase in the number of lavagable PMNs that was consistent with paraquat’s known pathophysiologic response in the lung. Paraquat was employed to illustrate
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how BALF analysis, with the aid of cytograv, can allow one to quantitate a pneumotoxicant’s ability to generate an inflammatory response. In summary, the cytograv method provided an easy and economical alternative to commercial cytocentrifuges. The cytograv method has many advantages over both cytocentrifuges and other sedimentation devices previously reported. These combined features of the cytograv should provide a valuable tool for investigators who deal with cytology of biological fluids, cell isolation, and identifications. The authors thank Dr. Vuokko Kinnula, Neilson for their advice and able technical supported, in part, by PO1 HL 31992.
Kathy Evans, assistance. This
and Bob study was
of lung lesions produced by ingested paraquat on mouse lung. Lab Invest
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