Abstract
A technique to standardise the analysis of cellular and non-cellular components in epithelial lining fluid (ELF) collected during saline lavage of pulmonary and pleural cavities was developed using the urea dilution method. Bronchoalveolar lavage (BAL) and pleural lavage (PL) fluids were collected from 12 clinically healthy cats. Total and differential cell counts in BAL fluid were within normal ranges for the cat, while cell counts in PL fluid were assumed to be normal based on clinical health during examination, auscultation and lactate dehydrogenase (LDH) activities being comparable with other species. The major clinical implication of this study was that nucleated cell counts within feline ELF could not be predicted from analysis of lavage fluid which suggests that calculation of the proportion of ELF in lavage fluid by the urea dilution method may be necessary to avoid misdiagnosis of health or disease in pulmonary or pleural cavities.
Bronchoalveolar lavage (BAL) is a relatively safe and easy technique to investigate disease processes deep in the lung (Hawkins et al 1994, Vail et al 1995). Epithelial lining fluid (ELF) from the alveoli and airways can be diagnostic for pulmonary diseases (Mayer et al 1990, Padrid 1991, Padrid et al 1991, Mills et al 1996). However, considerable variation has been reported in total cell counts and concentration of biochemical markers, such as lactate dehydrogenase (LDH) and glucose, which is partially due to variable recovery of ELF in the lavaged saline (Rennard et al 1986, McGorum et al 1993b). A method to account for this dilution of ELF using endogenous urea has been reported in humans (Rennard et al 1986) and the horse (McGorum et al 1993a, Mills et al 1996), but not the cat. A comparison between urea and inulin has also been performed in healthy and heaves-affected horses (Kirschvink et al 2001).
One remarkable aspect of differential cell counts in feline BAL samples is the high number of eosinophils (Padrid et al 1991, Hawkins et al 1994) which, in other species may be associated with parasitic infection or hypersensitivity within the respiratory tract (Kay 1985, Halliwell and Gorman 1989). Cell counts from BAL collection generally correlate well with histological examination of the lung (Hunninghake et al 1981, Padrid et al 1991) and it would be interesting to determine if eosinophil counts are comparatively high on the ‘other’ surface of the lung, the pleural surface. Little is known about normal cell counts in the pleural fluid of any species, although eosinophilia in pleural effusions was associated with neoplasia in the dog (Phillips and Schaer 1988, Cowgill and Neel 2003) and with idiopathic pleural effusions in humans (Rubins and Rubins, 1996, Martinez-Garcia et al 2000).
In this study, we collected BAL and pleural lavage (PL) fluid from 12 clinically healthy cats and adjusted cell counts and biochemical markers following correction for dilution using urea concentration. One of the problems associated with bronchoalveolar lavage is the highly variable recovery of ELF, while the use of urea adjusted cell counts and biochemical markers may eliminate some of the difficulty in interpretation by standardising ELF recovery in the comparatively large volumes of lavage fluid.
Materials and methods
Animals
Twelve mixed breed cats, aged between 4 months and 8 years, were used in this study. These cats had been selected from animals presented to the School of Veterinary Science for euthanasia from a local animal pound. Cats that were included in this study were clinically healthy, in good body condition and with no obvious respiratory disease on auscultation. This study had been approved by the Animal Ethics Committee of the University of Queensland (SVS/579/03).
Study design
The cats were sedated using acepromazine (0.1 mg/kg administered by subcutaneous injection 1 h earlier) and then euthanasia was performed using an intravenous overdose of sodium pentobarbitone (Lethabarb; Virbac, Australia). A 2.6 mm (8 Fr) dog urinary catheter (Arnolds, SIMS Portex, UK) was then immediately advanced via the trachea until it wedged in the left or right primary bronchus. Pilot studies had shown that there were no differences in cell counts between samples collected from one lobe of a cat deeply anaesthetised with intravenous sodium pentobarbitone compared with samples collected from the corresponding lobe on the opposite side immediately post mortem. Three 5 ml aliquots of warmed (25°C) sterile saline were flushed down the catheter, with immediate suction used to withdraw each flush before the subsequent aliquot was instilled. The lavage samples were combined in a sterile plastic vial and gently mixed before storage on ice. The abdominal cavity was opened and a blood sample withdrawn from the caudal vena cava. A small incision was then made through the seventh intercostal space at the level of the costo-chondral junction. Warm (25°C) sterile saline (10 ml) was syringed into the inter-pleural space and the cat was rocked gently for 30 s. The lavage fluid was then withdrawn by gentle suction and placed on ice. The lung was examined immediately after pleural lavage and the samples were discarded if there was any evidence of gross pathological changes.
BAL and pleural fluid analysis
BAL and PL fluid samples were analysed within 1 h of collection. Cell counts were performed on well-mixed undiluted fluid using an Improved Neubauer Haemocytometer (Assistant, Germany). Fluid samples then underwent cytocentrifugation (Shandon Cytospin 4, Theamo Shandon, Cheshire, UK) and differential cell counts prepared from smears after staining with Wright's stain (Hema-Tek 1000 Automatic Stainer, Ames, USA). Urea and lactate dehydrogenase (LDH) in BAL and pleural fluids (higher volumes (20 μl instead of 10 μl) of lavage fluid were used to account for lower analyte concentration) and in plasma were measured using an Olympus AU400 automatic analyser (Olympus, Tokyo, Japan). To facilitate the comparison between lavage fluid and the fluid lining the pleura and airways, the fluid lining the pleural membranes is referred to as ELF, although the authors recognise that the outermost layer of the pleura consists of a pavement layer of flat mesothelial cells overlying the epithelium.
Lung histopathology
Lung tissue specimens were collected from eight of the 12 cats in this study and immediately immersed in neutral buffered 10% formalin. These samples were processed by routine methods and embedded in paraffin. Specimens were sectioned to a thickness of 5 μm and stained with haematoxylin and eosin before independent interpretation.
Data analysis
The plasma:BAL fluid and plasma:PL fluid urea ratios were calculated for each sample. The product of these ratios and the cell count in BAL or PL fluid, respectively, yielded the urea adjusted cell in each fluid (McGorum et al 1993b). The proportion of ELF in BAL and PL fluid samples was calculated from the formula: BAL (or PL) fluid urea concentration×100/plasma urea concentration (Rennard et al 1986).
Regression analysis of the cell counts in ELF before and after correction for dilution was meaningless because, although all values increased, the relative amount of increase was related to the volume of ELF recovered and not the specific cell count. To illustrate this point, a relative change (BAL or PL cell count/ELF cell count) was calculated for each total nucleated cell count in the BAL and PL fluids, reflecting that cell counts increased substantially, but the corrected cell count could not be predicted from cell counts in the BAL or PL fluid. A box plot of log10 relative change was drawn for each fluid. All data are represented as mean±SD.
Results
Histopathological analysis of lung tissue from eight of the cats in this study confirmed clinical and cytological diagnosis of normal tissue. There was evidence of congestion, mild atelectasis and some focal emphysema, but there was no inflammatory response or infiltrates in any of the tissues examined. The recovery of saline from BAL and PL was 73.1±6.4% and 90.5±3.6%, respectively. The proportion of ELF in BAL fluid and PL fluid was 4.6±3.2% and 16.3±7.1%, respectively. The cell counts in BAL or PL fluids, and the urea adjusted cell counts, are listed in Table 1 for BAL fluid and Table 2 for PL fluid. Box plots of log10 relative change for nucleated cell counts in BAL and PL fluids are shown in Fig 1. The activity of LDH in BAL and PL fluids was 1.9; 0.1, 13.0; 0.95, 4.5 U/l (median; range; interquartile range) and 10.0; 2.0, 34.0; 6.0, 13.0 U/l, respectively, while the urea adjusted values were 114.6; 3.3, 253.5; 32.7, 160.2 and 52.6; 10.1, 136.5; 36.1, 119.6 U/l, respectively.
Box plot of log10 relative change of nucleated cell counts between BAL or PL fluid and the respective ELF for 12 cats (mean±SD). The line within each box represents the median value; the upper and lower lines of the box represent the 75th and 25th centiles, respectively, and the upper and lower whiskers represent the 90th and 10th centiles, respectively.
Cell populations in bronchoalveolar lavage (BAL) fluid and epithelial lining fluid (ELF; after adjustment for urea concentration) collected from 12 cats (median, interquartile range)
Cell populations in pleural lavage (PL) fluid and epithelial lining fluid (ELF; after adjustment for urea concentration) collected from 12 cats (median, interquartile range)
Discussion
In this study, we have presented a technique to reduce the major source of error when performing BAL by directly calculating how much ELF is contained in the lavage sample. There are a number of guidelines that have been suggested to reduce the variability in BAL cytology caused by the technique itself, including using different lavage volumes and residence times (time between instilling and then withdrawing the saline) and analysing each aliquot separately (Hawkins et al 1990, 1994, Vail et al 1995). Urea diffuses freely throughout the body, including the alveolar wall (Taylor et al 1965), and has been used in other species to accurately calculate the concentration of cells and biochemical markers in ELF (McGorum et al 1993a, Rennard et al 1986, Mills et al 1996). We were able to use this principle to calculate a corrected cell count which reflects actual cell concentrations within the ELF of the cat.
The BAL cell counts from the cats in this study were within the range of previously reported values for the normal cat (Hawkins et al 1990, Padrid et al 1991). As expected, the ELF cell count was substantially higher owing to the relatively small proportion (4.6±3.2%) of ELF in BAL fluid. We were particularly interested in the relationship between the raw and adjusted cell counts because a direct linear relationship would suggest that ELF cell counts could be predicted from BAL cell counts. However, simple correlation analysis was unsuitable for data analysis because the values are not independent, while scatter plots of individual BAL vs ELF cell counts revealed that BAL cell counts could not be used to predict ELF cell counts. This was demonstrated graphically in the box plot where the relative change for nucleated cell counts was highly variable.
The differential cell count as a percentage of the total nucleated cell count was, as expected, unaffected. The differential percentage of nucleated cells may attract closer scrutiny when evaluating BAL fluid because of the marked variability of total cell counts in normal samples (Vail et al 1995). An increase in the relative proportion of a particular cell type is frequently associated with specific disease processes. For example, an increase in eosinophils reflects parasitic infection or hypersensitivity reaction within the respiratory tract (Kay 1985, Halliwell and Gorman 1989). A variation in the proportion of eosinophils up to 25% or more may be considered normal in the cat and not be associated with any gross or histological pathology in the lung (Padrid 1991). It should be noted, however, that actual cell numbers within ELF may increase from the contribution of more than one cell type and, unless absolute cell counts are known, may obscure disease processes, particularly when relying on BAL fluid analysis with poor ELF recovery. This study has found highly variable nucleated cell counts in the BAL fluid from healthy cats, as have other previously reported studies (Padrid et al 1991). However, as nucleated cell counts in the ELF are also highly variable, it seems unlikely that variable dilution rates in BAL fluid samples alone can account for this.
The use of urea as an endogenous marker of dilution is not without potential error because the technique assumes that the concentrations of urea across plasma and epithelial lining fluid are constant over time. A prolonged BAL procedure (>2 min) may, however, permit the movement of urea into the air spaces (Effros et al 1990, 1992, Ward et al 1992, 1999) and thereby overestimate ELF volumes (and reduce corrected cell counts). A rapid and consistent technique would reduce this source of variability and also reduce differences between cell counts reported as normal values in the literature. Importantly, increased permeability of the plasma to airway barrier for urea may be expected during pathological conditions (Marcy et al 1987), although urea is known to equilibrate rapidly between ELF and plasma in normal tissue (Rennard et al 1986, Ward et al 1992). In this study, we used a standard and rapid BAL technique to minimise potential error in a sample that may be collected during routine clinical investigation of airway disorders without using exogenous markers, such as inulin (Kirschvink et al 2001) or radiolabelled markers (Effros et al 1992, Ward et al 1992). The most clinically relevant finding did not pertain to absolute cell counts within ELF but to the fact that absolute cell counts cannot be predicted from BAL cell counts. It should be noted, however, that ELF volume would normally increase during bronchopulmonary disease (Marcy et al 1987, Hawkins et al 1990), and the recovery of ELF during lavage procedures could increase dramatically or, particularly in pleural spaces, even exceed the volume of lavage fluid.
The potential for error using urea as an endogenous marker is less evident in pleural fluid, particularly in healthy pleural cavities (Noppen et al 2000). We have reported, for the first time, cell counts following PL in the normal cat immediately post mortem. Similar to BAL fluid, there was no clear relationship between cell counts in pleural fluid and pleural ELF. Pleural effusions are a relatively common medical condition in cats (Noone 1985, Tyler and Cowell 1989) and specific cell types within pleural effusion fluid, such as eosinophils, have been reported in association with neoplasia in the dog (Phillips and Schaer 1988, Cowgill and Neel 2003) and with idiopathic pleural effusions in humans (Rubins and Rubins 1996, Martinez-Garcia et al 2000). However, based on the findings of a consistently low proportion of eosinophils (<2%) in feline pleural ELF in this study, a relative increase in this proportion could be associated with disease processes if the cat is similar to other species, but further studies would be needed to verify this. The predominant cell in normal feline pleural fluid, as in BAL fluid, was the macrophage, with lymphocytes and neutrophils as other major cell types. Eosinophils comprised about 1% of nucleated cells in PL fluid, unlike BAL fluid, which suggests that large numbers of eosinophils in pleural effusions may be associated with pathological changes.
A parameter to assess tissue injury and pathological states in extra-cellular fluids, other than cell counts, is the activity of LDH (Smith et al 1988, Pusch et al 1997, Emad and Rezaian 1999). Here we report BAL LDH activity of 110.2±212.3 U/l which is compared with BAL fluid from healthy lambs (145 U/l; Pusch et al 1997) and humans (64 U/l; Cobben et al 1999). To the author's knowledge, reference values for LDH activity in normal feline PL fluid remain unreported, but we presume that our reported figure of LDH activity in pleural ELF (71.7±70.1 U/l) was representative of normal PL fluid as all cats were clinically healthy with no auscultatory abnormalities.
In conclusion, this study has shown that it is possible to estimate ELF cell counts and biochemical parameters in cats, as in other species, and this provides a useful basis for further studies of cats with bronchopulmonary and/or pleural disease to see if quantitative assessments of these parameters can provide more valuable data. Cell counts in lavage fluid are not reflective of cell counts in ELF and may lead to incorrect assumptions of health or disease. Estimation of ELF fraction in lavage fluid using standardised lavage technique and correction for urea dilution permit a more accurate measure of cellular and non-cellular components of ELF.
Footnotes
Acknowledgements
The authors would like to thank Brian Bynon for performing the biochemical and cytological analyses of pleural and bronchoalveolar lavage fluids and Dr Brett Stone for interpretation of histopathology. This study was made possible with the generous support of the John and Mary Kibble Trust.