Rewash Bronchoalveolar Lavage1 •2

DAVID E. GRIFFITH, BARRY T. PETERSON, and MICHAEL L. COLLINS

Introduction SUMMARY A significant limitation of standard bronchoalveolar lavage (SAL) technique Is the In-

Standard bronchoalveolar lavage (BAL) ability to measure or calculate epithelial lining fluid (ELF) volume and, therefore, In vivo concentratechnique involves instillations of physitions of substances In the ELF. We evaluated a new rewash BAL procedure with the radlolabeled ologic saline into the lower respiratory tracer technetium pertechnetate (tlmTcO..-) that theoretically should be Immune to even exaggertract followed by aspiration and recovated fluid shifts during BAL. To test this theory, we measured ELF volume In control sheep using ery of the fluid. The instillation-aspiraIsosmotic (280 mosm/L) hypoosmotlc (140mosm/L) and hyperosmotlc (570 mosm/L) SAL solutions to Induce exaggerated fluid shifts during the lavage procedure. The mean ELF volume of the lavaged tion sequence is then repeated five to six lung segment was not significantly different for the three solutions (Isosmotic, 1.7 ± 0.8ml; hypooatimes. Because the fluid recovered by motlc,1.1 ± 1.2 ml; hyperosmotlc, 2.1 ± 1.8 ml). The slope of the 88mTcO..- disappearance curve, BAL is a variable mixture of saline, epihowever, was significantly steeper for the hyperosmotlc solution (- 0.40 ± 0.04%/mln) compared theliallining fluid (ELF), ELF compowith the other solutions (Isosmotic, -0.14 ± .01%/mln; hypoosmotlc, -0.12 ± O.07%/mln).Calcunents, and extra-alveolar fluid, it has been lation of ELF volume using sodium as an endogenous tracer gave consistently smaller values with impossibleto convertmeasurements made each of the mannitol solutions (Isosmotic, 0.21 ± 0.30 ml; hypoosmotlc, 0.02 ± 0.03 ml; hyperoaon the collected fluid into estimates of motlc, 0.18 ± 0.18 ml). The failure of sodium to provide accurate estimates of the ELF volume may actual in situ concentrations in the ELF. be due to complicated sodium movement In the lung and errors In our aaaumption of the Initial The inability to estimate the concentraconcentration of sodium In the ELF fluid. We conclude that the rewash SAL technique with "mtcO..tions of ELF components is perhaps the gives values of ELF volume that are not significantly affected by even exaggeration of the fluid flux that Invariably accompanies BAL. AM REV RESPIR DIS 1991; 144:151-155 single most important shortcoming of this otherwise very useful procedure. To circumvent the problem of ELF dilution during BAL, efforts have been The problem of calculating ELF vol- or influx of fluid into the alveolar space made to "normalize" biochemical measurements to a particular molecule such ume using a tracer with standard BAL during the lavageare minimized by extrapas albumin. This sort of analysis pro- technique is probably not limited to urea. olating the tracer concentrations back to vides estimates of relative concentra- It may exist for any dilutional marker be- time zero when the lavage solution had tions in BAL but does not yield estimates cause of altered fluid dynamics in the al- mixed with the ELF but had not been of actual ELF volume or ELF solute veolar space during BAL. Kelly and co- affected by loss of the tracer from the workers (3) determined that there is a sig- alveolar spaceor by altered fluid dynamics. concentrations. An alternative effort has been made nificant bidirectional flux of fluid into In initial experiments, the rewash lato calculate a dilution factor that would and out of the alveolar space during stan- vage technique gave reasonable estimates allow the ELF volume to be calculated dard BAL so that, of the total aspirated for ELF volume and ELF protein confrom the lavage fluid concentration of volume of recovered BAL fluid, approx- centrations in normal sheep and sheep a "tracer" substance. Rennard and co- imately 40070 is water that has entered the with oleic acid lung injury (4). Because workers (1)have suggested the measure- alveolar space from the bloodstream dur- this ELF volume is calculated from the ment of BAL urea concentration as a use- ing the procedure. time zero intercept and not from the slope Peterson and coworkers (4) recently in- of the 99mTc04- disappearance curve, we ful method to determine this dilution factor. The low molecular weight and high troduced a new "rewash"BAL lavagepro- hypothesized that the rewash BAL techdiffusibility of urea support the assump- cedure to measure ELF volumes and ELF nique would be immune to even exaggertion that the ELF urea concentration is solute concentrations in sheep lungs. This ated shifts in fluid in the alveolar space in equilibrium with the plasma urea con- method is analogous to a rebreathing during lavageand would therefore be usecentration, which is an important prem- technique previously described for mea- ful under a wide range of physiologic ise for ELF volume estimation with urea. suring lung tissue volume (5, 6). With the conditions. To test this hypothesis, we This equilibrium is disturbed, however, bronchoscope wedged into a distal air- performed rewash BAL on normal sheep with the instillation of normal saline in- way,a lung segment is lavaged with four using isosmotic, hypoosmotic, and to the lower respiratory tract during BAL cycles of instillation and aspiration of a hyperosmotic solutions to promote exbecause the addition of saline creates a single aliquot of the lavage solution con.concentration gradient for urea between taining a radioactive tracer, technetium (Received in original form November 20, 1990) plasma and the alveolar space. Marcy and pertechnetate (99mTc04-). The tracer coworkers (2) have demonstrated that this concentration is measured with each ap1 From the Departments of Medicine and Physgradient causes a progressive increase in plication of the fluid aliquot and plotted iology, The University of Texas Health Center at Tyler, Texas. the concentration of urea with each se- against time. This plot represents a dis- Tyler, 2 Correspondence and requests for reprints appearance curve for 99mTc04-. Potential quential instillation of lavage fluid, should be addressed to David E. Griffith, M.D., resulting in an overestimation of the cal- errors in calculating ELF volume because The University of TexasHealth Center at Tyler,P.O. of changing concentrations of the tracer Box 2003, Tyler, TX 75710. culated ELF volume. 151

152

GRIFFITH, PETERSON, AND COLLINS

aggerated fluid shifts into and out of the alveolar space. We also hypothesized that the rewash technique might not require an exogenous radiolabeled tracer and could be performed using an endogenous biochemical tracer. That is, the rewash procedure might be equally applicable for a tracer "washing in" as for one "washing out." To test this hypothesis, we performed rewash BAL on normal sheep with sodium-free mannitol solutions of various osmolality. We then calculated ELF volume using sodium concentrations recovered in the BAL fluid. Methods Rewash Lavage and ELF Calculation All studies were approved in advance by the Animal Research Committee of the University of Texas Health Center at TYler. Each sheep was anesthetized with 0.5 to 1.0g pentothal sodium, intubated, placed in a prone position, and ventilated with 1 to 2070 halothane in room air. A 5-ml blood sample was then collected from the external jugular vein. A stock lavage solution was prepared on the day of the' experiment and consisted of either isosmotic saline (280 mosm/L), isosmotic mannitol (280 mosm/L), hypoosmotic mannitol (140mosm/L), or hyperosmotic mannitol (570mosm/L). The mannitol solutions were prepared with pharmaceuticalgrade mannitol (Sigma Chemical, St. Louis, MO). Osmolality of each solution was confirmed with an osmometer. 1Wohundred milliliters of the stock lavage solution weremixed with approximately 0.5 mCi 99mTc04-. A fiberoptic bronchoscope 6 mm in diameter was introduced into the tracheobronchial tree through an adapter on the endotracheal tube to allow simultaneous mechanical ventilation of the sheep during the procedure. We wedged the tip of the bronchoscope into a distal airwayof either the right or left lung. A syringe with 50 ml of the stock lavage solution was attached to the suction channel of the bronchoscope via a three-way stopcock (figure 1). The lavage solution was emptied into the lung segment and then aspirated back into the original syringe. Wequickly collected a 0.7-ml sample of the aspirated solution with a l-ml syringe attached by the sidearm of the three-way stopcock. The remainder of the aspirated fluid was then flushed back into the lungs. This procedure was repeated a total of four times. A foot pedal was used to activate a marker on a strip chart recorder to mark the time at which the lavage began, the time at which the syringe was first emptied, and the time at which each sample was collected. After the fourth 0.7-ml sample was collected, the bronchoscope was removed from the sheep and rinsed with a solution of appropriate osmolality. Approximately 2 to 4 min later, a second rewash lavage was performed with the bronchoscope

-.

1 ml sample syringe

,CD ®;® ~

o

60

120

Seconds

wedged into a different subsegmental bronchus in the same lung. A third subsegmental bronchus was used for the final lavage. Once all three lavages were complete, we measured the radioactivity in four 0.5-ml samples of the stock lavage solution and in 0.5-ml samples of each collected lavage fraction. The standard deviation of the measurement of the activity in the stock lavage solution was usually 0.5070 of the mean and always less than 1070. The final (fourth) lavagefraction from each rewash procedure was centrifuged at 500 g for 5 min to separate cellular and noncellular elements. The supernatant was then frozen at -70 0 C for protein and sodium analysis. Total BAL cell counts were obtained with a Coulter counter (Coulter Electronics, Hialeah, FL). Differential cell counts were determined from cytocentrifuge preparations stained with a modified Wright-Giemsa stain. Total numbers of BAL macrophages, neutrophils, lymphocytes, and eosinophils were obtained by multiplying the cell differential for each by the total white cell count obtained. The ELF volume calculation is described in detail elsewhere (4). Briefly, we divided the specific radioactivity (cpm/ml) in each fraction by the specific radioactivity in the stock lavage solution and plotted this ratio (percent 99mTc04- remaining) against the time each fraction was collected. Time zero was taken to be the time one half of lavage fluid entered the lungs (approximately 3 to 4 s after the beginning of the lavage procedure). A least squares regression analysis selected values for A o and k to fit the data to a single exponential decay equation: A(t)

= Ao x

e-kt

(1)

Fig. 1. The rewash BAL technique was performed by instilling 50 ml of the lavage solution containing saline and technetium pertechnetate tracer (TeO..-) into one segment of the lungs. The so. lution was drawn back into the original syringe, and a 0.7-ml sample was collected in the 1-ml syringe on the side. The fluid was then reinstilled into the segment, and the cycle was repeated a total of four times. The time at which each sample was collected was recorded on a strip chart recorder (Reprinted with permission from Am Rev Respir Dis 1990; 141:315.)

where A(t) is the concentration of 99mTc04in the lavage fluid-ELF mixture at any time (t) expressed as a percent of the initial concentration in the lavage syringe, A o is the time zero intercept of A(t), and k is the clearance rate constant in min- t • The analysis also provided a root-mean-square (RMS) estimate of the error in the curve-fit as a percent of the intercept value. A previous study with an analogous method showed that incomplete mixing in the lungs produced large RMS error. Because complete mixing is an important assumption in our model, lavages with large errors (> 1.0070) wereexcluded from the study (equation 1) (6). The value of A o as a percent of the initial concentration in the stock lavagesolution was assumed to be the concentration of the 99mTc04- in the mixture of the 50 ml of lavage fluid and the unknown volume of ELF at a time at which mixing between the lavage fluid and the ELF was complete but no 99mTc04- had left the air space and no fluid had entered the air spade from the interstitium. If none of the tracer is lost from the system and no fluid enters the lungs at time zero, then equating the total amount of the tracer in the system before arid after the mixing yields: 50 ml x 100070 = (50 ml + ELF volume)

x Ao

(2)

Solving for the ELF volume gives: ELF volume (ml) = (100070 - A o) 50 ml x A

(3)

o

which we used to calculate the ELF volume in the lavage segment.

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REWASH BRONCHOALVEOLAR LAVAGE

Sodium Measurement and Calculation of ELF Sodium analyses wereperformed on an atomic absorption spectrophotometer (Model IL 551; Instrumentation Laboratories, Lexington, MA). The lavage samples were diluted to bring the sodium concentration into the linear range of the spectrophotometer (zero to 3 ppm). First, the lavagesamples werediluted 1:100 by adding 50.5 J.11 of the sample to 5.0 ml of 18 M ohm water. From this 1:100 dilution, four 1:10serial dilutions were made by using 500 IJI of the previous dilution. Each dilution sample was read five times by the spectrophotometer. The standard error was between 0.2 and 0.8OJo. To insure there was no sodium contamination from the test tubes, samples were prepared in tubes that were sodium-free (Nonick, Copenhagen, Denmark). The standard curve was made from dilutions of atomic spectral standard (Fisher Scientific, Springfield, NJ). The sodium concentration in each lavage sample was divided by the measured sodium concentration in the plasma sample. We assumed that before the lavage began, the sodium concentrations in the plasma and the ELF were the same. Therefore, the ELF volume using BAL sodium was calculated as: ELF volume

=

50 ml x [Na+]To 1 - [Na'[n,

(4)

where [Na+ Ire is the extrapolated time zero concentration of Na in the ELF.

Determination of Protein Concentrations The total protein concentrations in the collected lavage fluids and plasma samples were determined by a commercially available colorimetric assay (Bio-Rad, Richmond, CA). The protein concentration in the ELF wascalculated as: ELF protein concentration (mg/ml) = (50 + ELF) (5) [P] (mg/ml x ELF where [P] is the measured protein concentration in the last (fourth) collected lavage fraction in mg/ml and ELF is the volume of the ELF in ml calculated as described above (4).

Statistical Analysis All data are reported as means ± so. Differences among all values in the groups were compared first by using a one-way analysis of variance on all groups and then by using the modified t test for determining the t statistic for pairs of groups. The critical t value for significance was determined by the Bonferroni method (7). Results

ELF Volumes Sixty-three rewash lavageswere performed in normal sheep. Sixteen rewash lavages (25070) were excluded from analysis because of greater than 1.0070 error in curve-

TABLE 1 EPITHELIAL LINING FLUID (ELF) ANALYSIS UTILIZING 88mTcO..-* Isosmotic Mannitol

Saline 12 1.7 ± 0.8

Lavages, n ELF volume, ml Rate of change in tracer concentration, %/s ELF protein concentration, % of plasma value

Hypoosmotic Mannitol

12 1.8 ± 0.8

9 1.1 ± 1.2

-0.10 ± 0.05

-0.14 ± 0.07

-0.12 ± 0.07

24.0 ± 12.0

30.0 ± 18.0

27.0 ± 15.0

Hyperosmotic Mannitol 7 2.1 ± 1.6 - 0.40 ± O.04t 14.0 ± 5.0t

* Values are mean ± SO. with saline, isosmotic mannitol, and hypoosmotic mannitol.

t p < 0.05 compared

fit as described in METHODS. ELF volume determinations using 99mTc04- are summarized in table 1.The ELF volumes calculated with isosmotic saline and isosmotic mannitol as the lavage solution were 1.7 ± 0.8 and 1.8 ± 0.8 ml, respectively. These values are almost identical to the value previously determined using the rewash technique with saline in normal sheep (4). The slopes of the 99mTc04decay curves for isosmotic saline and mannitol were the same (see table 1 and figure 2). The ELF volume determined using hypoosmotic mannitol (140mosm/ L) was 1.1 ± 1.2 ml. Although this value is less than that determined with the isosmotic solutions, the difference did not reach statistical significance. The slope of the disappearance curve for the hypoosmotic mannitol was also smaller but not significantly different from those generated with the isosmotic solutions (table 1). The ELF volume determined with hyperosmotic saline was 2.1 ± 1.6ml (table 1), which was not significantly differ-

100

.S

.;d 8 ~

ent from that determined using isosmotic solutions or the hypoosmotic solution. However, the slope of the 99mTc04- disappearance curve had a significantly greater slope than did either the isosmotic or the hypoosmotic solutions (figure 3). Despite the 3-fold increase in the slope of the 99mTc04- disappearance curve, the time zero intercept and thus the ELF volume calculation remained the same as that observed with isosmotic mannitol. The ELF volumes determined using the sodium concentration in the recovered BAL fluid are shown in table 2. For each solution used (isosmotic, hypoosmotic, and hyperosmotic mannitol), the calculated ELF volume was significantly lower than that determined with 99mTc04-.

Protein Concentrations The protein concentration in the plasma was 95 ± 26 mg/ml. The mean ELF protein concentrations, expressed as a percent of the plasma value, determined with 99mTc04- are summarized in table 1. There were no statistically significant differences in ELF protein concentration among the isosmotic saline, isosmotic

90

100

tlIl

.S

80

.;d 8 ~

70

90

•

80

• 60

-r--"'---'-~--'--r--'T---r---.--.--....----r----r--'--"T""""""""""T---r-----.

°

10

20

30

40

50

60

70

80

Time (sec)

Fig. 2. The fraction of tracer (88mTcO..-) remaining in each lavage fraction was plotted against time, and monoexponentialline of best fit was extrapolated back to zero time. This figure shows the data and the calculated ELF volume from a representative lavage with isosmotic mannitol. The volume of the ELF was calculated from the intercept by equation 3 in the text. The slopes of the tracer disappearance curves for isosmotic and hypoosmotic mannitol were not significantly different from that of saline. Intercept = 96.3%; slope = -0.0690/o/s; ELF Vol. 1.9 ml.

=

60 ~...---,-----r----r---r--'T--.-~~r---r----r--.---r---'1--'---' o 10 20 30 40 50 60 70 80

Time (sec)

Fig. 3. This figure shows the data and the calculated ELF volume from a representative lavage with hyperosmotic mannitol. The slope of the tracer disappearance curve is significantly steeper than with the other lavage solutions, although the zero time intercept and subsequent ELF calculation are not significantly affected. Intercept = 96.1%; slope = -0.345%15; ELF Vol. = 2.0 ml.

154

GRIFFITH, PETERSON, AND COLLINS

TABLE 2 EPITHELIAL LINING FLUID (ELF) VOLUME DETERMINATIONS UTILIZING SODIUM* Isosmotic Mannitol Lavages, n ELF volume, ml Rate of change in sodium concentration

Hypoosmotic Mannitol

Hyperosmotic Mannitol

8

5

0.22 ± 0.29

0.02 ± 0.03

7 0.18 ± 0.18

0.05 ± 0.03

0.07 ± 0.01

0.09 ± 0.03

* Values are mean ± SO.

TABLE 3 COMPARISON OF REWASH AND STANDARD BAL CELL RECOVERY* Rewash BALt Lavages, n Total cells, x 105/ml AM, o~ PMN,% Lymphs, o~ EOS,% Volume of lavage fluid returned Lavage fluid returned, o~

31 4.5 ± 80.4 ± 0.8 ± 15.6 ± 3.2 ± 11.3 ± 22.6

0.5 1.0 0.2 0.9 0.5 5.9

*

Standard BAL

p Value

27 1.8 ± 0.1 83.9 ± 1.9 7.4 ± 1.3 7.6 ± 1.1 1.1 ± 0.5 63.0 ± 13.0 63.0

< 0.01 > 0.05 < 0.01 < 0.01 < 0.01 < 0.01

Definition of abbreviations: AM = alveolar macrophages; PMN = neutrophils; Lymphs = lymphocytes; EOS = eosinophils. * Values are mean ± SO. isosmotic mannitol, hypoosmotic mannitol, hyperosmotic mannitol. :I: Saline.

t Saline,

Discussion Theoretically, the rewash BAL technique originally described by Peterson and coworkers (4) should provide estimates of ELF volume that are immune to the fluid shifts that invariably accompany BAL. In the present study, we examined whether exaggerated fluid shifts induced by hypoosmolar or hyperosmolar lavage fluCell Data id significantly affected ELF volume calWe compared rewash BAL cell recovery culations. As predicted, the exaggerated data with standard BAL cell recovery in influx of water into the alveolar space normal sheep from a previous study induced by the hyperosmolar lavage solution accentuated the fall in 99mTc04reported by our laboratory (8). The standard BAL protocol consisted of five2o-ml concentration. However, neither the time zero intercept nor the calculated ELF volaliquots of saline performed in each animal. The data are summarized in table ume were significantly affected. The hyperosmotic solution did not alter the 3. Rewash lavage recovered higher concentrations of cells than did standard ELF calculation despite a significant change in the slope of tracer disappearlavage (4.5 ± 0.5 versus 1.8 ± 0.1 x 105 cells/ml, p < 0.01), although the total ance. Therefore, the assumptions of adequate mixing (Le., when the RMS estinumber of cells recovered was less with the rewash method because of the smaller mate of error is less than 1070 and when volume of return. The cell differential there is monoexponential disappearance counts were also significantly different of technecium) appear to be reasonable. The protein concentration was calcubetween the two methods. Rewash lavage recovered a significantly lower percent- . lated from the fourth (and final) aspiage of neutrophils than did standard la- rated lavage aliquot, after shifts in the vage (0.8 ± 0.2 versus 7.4 ± 1.3070, p < fluid had time to manifest themselves. 0.01) but a significantly higher percent- Therefore, the calculated protein concenage of lymphocytes (15.6 ± 0.9 versus trations would be falsely low if fluid 7.6 ± 1.1, P < 0.01) and eosinophils (3.2 enters the air spaces during the lavage. The dilution of proteins also supports the ± 0.5 versus 1.1 ± 0.5070, p < 0.01).

mannitol, and hypoosmotic mannitol groups. There was, however, a statistically significant decrease in the calculated ELF protein concentration with hyperosmotic mannitol as the lavage solution. This finding is consistent with dilution of ELF proteins by fluid influx caused by the hyperosmotic solution.

interpretation that the accelerated decline in 99mTc04- concentration with the hyperosmolar solution was due to fluid influx into the alveolar space as opposed to accelerated loss of the tracer. This potential source of error in protein measurement had been recognized previously (4). The ELF protein concentration may, therefore, be slightly underestimated, even under relatively stable conditions using the fourth rewash specimen for protein measurement. Rewash BAL using 570 mosm/L mannitol (2 times normal serum osmolality) produces artificially rapid and dramatic fluid shifts that would be very unusual physiologically. For instance, in the presence of extreme hyponatremia, physiologic saline introduced by BAL might be, at most, 1.3 times serum osmolality. Therefore, the observed 40070 underestimation of protein concentration could be considered beyond the boundary of conditions seen clinically. The hypoosmotic lavage solution gave a similar or slightly less steep slope of 99mTc04- disappearance than the isosmotic solutions. This observation is consistent with the expected opposing effect of the hypoosmotic solution on the movement of water into the alveolar space. Dse of sodium as an endogenous tracer gave consistently smaller calculated ELF volumes than did 99mTc04-. One of the primary differences between the 99mTc04- and sodium techniques is that the initial 99mTc04- concentration in the stock solution is measured, whereas the initial sodium concentration in ELF is not measured but assumed to be equal to that in the plasma. This assumption is based on limited data in an animal model (9). Recent measurements ofbronchial surface sodium concentration in humans indicate that this fluid may be hypotonic (10); therefore, it is possible that the ELF sodium concentration may be less than the plasma sodium concentration. If so, our calculations would underestimate the true ELF volume, which indeed occurred. Another problemwith the use of sodium is that its movement in the body may not be well described by our model of simple first-order 'kinetics. It may be subject to active transport and concentration, osmotic, ionic or electrochemical gradients that are difficult to model accurately during the lavage procedure. In this regard, 99mTc04- is a superior tracer because no assumptions about plasma 99mTc04- concentration compared with ELF 99mTc04- concentration are necessary. Further, 99mTc04- disapI

REWASH BRONCHOALVEOLAR LAVAGE

pearance appears to be well described by a simple model (4). Theoretically, the rewash technique could be used with a number of endogenous tracers, including urea. Urea may have some advantage over sodium with the rewash technique because of its diffusibility across physiologic membranes. However, 99mTc04- appears to be superior to endogenous tracers because the initial concentration is measured, not assumed. Additionally, no further assumptions are required about altered physiology. It is possible, however, that some as yet unrecognized endogenous tracer could be used with the rewash technique to provide accurate estimates of ELF volume. The cellrecoverydata with rewashBAL are significantly different than with standard BAL. The reasons are not immediately clear, but these differences may reflect different efficiencies in lavaging the alveolar spaces. For example, the lower neutrophil fraction obtained with the rewash lavage technique may be due to more complete sampling of the alveolar ELF fluid and less sampling of airway fluid, which has been shown to have higher neutrophil populations in humans (11). However, we know of no histologic data of resident cellpopulations in sheep lung; consequently, wecannot say which of the two lavage techniques provides a more accurate sampling of cells in the alveolar space. Although cell concentrations are higher with rewash BAL than with standard BAL, the lower volume of return means the rewash technique will recover fewer

155

total cells per lavage. This relative inadequacy is largely offset by the fact that three rewash lavagesare easily performed in one lung over a short period of time, thus increasing the total harvest of cells substantially. One problem with the rewash BAL procedure is that the ELF volume is very small compared with the instilled lavage fluid volume; therefore, small errors in the time zero extrapolation value may be magnified to produce large changes in the calculated ELF volume. This problem is addressed by using strict criteria for 99mTc04- disappearance curve-fit as a percent of the intercept value. However, this effort to improve data accuracy introduces another problem, particularly for human subjects. Even if multiple segments are lavaged, it is possible that some of the 99mTc04- disappearance curves would not meet acceptance criteria, thus eliminating those lavages from consideration of ELF calculation. Fortunately, the fluid and cells could still be used, however, in a manner similar to standard lavage technique. In summary, we have shown that the rewash BAL technique for estimating ELF volumes remains accurate in a wide variety of physiologic conditions, including use of saline or mannitol solutions with dramatic shifts in lung fluid. This supports the validity of the mathematical model used to calculate ELF volumes using the rewash technique. Acknowledgment The writers thank Sandy Carlson, KayChampion, and Glenda Battles for their consider-

able assistance in the preparation of the manuscript. References 1. Rennard SI, Basset G, Lecossier 0, et al. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol 1986; 60:532-8. 2. Marcy TW, Merrill WW, Rankin JA, Reynolds HY. Limitations of using urea to quantify epitheliallining fluid recovered by bronchoalveolar lavage. Am Rev Respir Dis 1987; 135:1276-80. 3. Kelly CA, Fenwick JD, Corris PA, Fleetwood A, Hendrick OJ, Walters EH. Fluid dynamics during bronchoalveolar lavage. Am Rev Respir Dis 1988; 138:81-4. 4. Peterson BT, Idell S, MacArthur CA, Gray LD, Cohen AB. A modified bronchoalveolar lavage procedure that allows measurement of lung epithelial lining fluid volume. Am Rev Respir Dis 1990; 141:314-20. 5. Peterson BT, Petrini MF, Hyde RW, Schreiner BF. Pulmonary tissue volume in dogs during pulmonaryedema. J Appl Physiol1978; 44:782-95. 6. Petrini MF, Peterson BT, Hyde RW. Lung tissue volume and blood flow by rebreathing: theory. J Appl Physiol 1978; 44:795-802. 7. Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res 1980; 47:1-9. 8. Idell S, Peterson BT, Gonzalez KK, et ale Local abnormalities of coagulation and fibrinolysis and alveolar fibrin deposition in sheep with oleic acidinduced lung injury. Am Rev Respir Dis 1988; 138:1282-94. 9. Nielson OW. Electrolyte composition of pulmonary alveolar subphase in anesthetized rabbits. J Appl Physiol 1986; 60:972-9. 10. Wagner G, Church N, Gatzy JT, Boucher RC, Knowles MR. Airway surface liquid (ASL) composition in normal humans (abstract). Am Rev Respir Dis 1990; 141:AI06. 11. Thompson AB, Daughton D, Robbins RA, Ghafouri MA, Oehlerking M, Rennard SI. Intraluminal airway inflammation in chronic bronchitis. Am Rev Respir Dis 1989; 140:1527-37.