Lung Lavage

BAL Alveolar Hemorrhage

Abstract Bronchoalveolar lavage BAL is a useful tool in researches and in clinical medicine of lung diseases because the BAL fluid contains biochemical and cytological indicators of the cellular response to infection, drugs, or toxicants.

Abstract Bronchoalveolar lavage BAL is a useful tool in researches and in clinical medicine of lung diseases because the BAL fluid contains biochemical and cytological indicators of the cellular response to infection, drugs, or toxicants. However, the variability among laboratories regarding the technique and the processing of the BAL material limits clinical research. The aim of this study was to determine the suction frequency and lavage fraction number necessary to reduce the variability in lavage using male Sprague-Dawley rats.

We compared the total cell number and protein level of each lavage fraction and concluded that more cells and protein can be obtained by repetitive lavage with a suction frequency of 2 or 3 than by lavage with a single suction. These observations were confirmed in bleomycin-induced inflamed lungs of rats. We further compared the BAL data from the whole lobes with data from the right lobes and concluded that BAL data of the right lobes represented data of the whole lobes.

However, this conclusion can only be applied to general lung diseases. At the end, this study provides an insight into the technical or analytical problems of lavage study in vivo. BAL was first used to treat a patient with phosgene gas poisoning in Gee and Fick, This approach has now been extended to investigate the pathogenesis, diagnosis, and therapeutic management of lung diseases in humans Daniele et al.

In laboratory animals, Myrvik obtained alveolar macrophage from rabbit lung lavage to compare physiological and functional ability in Gee and Fick, Since then, BAL has been commonly analyzed for monitoring the therapeutic efficacy and toxicity in laboratory animals Tornling et al.

When the disease or toxicity is limited to the lung, some useful information can be obtained by BAL, whereas blood analysis does not always indicate the state of lung. BAL has been widely used for pre-clinical and clinical research because BAL fluid contains both biochemical and cytological indicators of cellular responses to infection, cancer, or inhaled drugs or toxicants Hunninghake et al.

This concept was supported by reports showing a reasonable correlation between the cellular features of open lung biopsies and the cell population derived from BAL Haslam et al. However, one of the main obstacles for general acceptance of BAL as a clinical or a research tool is the variability regarding the lavage technique and the processing of the BAL material among laboratories Cordeiro and Cemylyn-Jones, ; Crystal et al.

Many investigators, including European Respiratory Society, have tried to establish methods or guidelines for the measurement and standardization of BAL in humans Haslam and Baughman, ; Walters and Gardiner, ; Crystal et al. To reduce variability between each BAL trial, investigators recommend a standard introduction volume of lavage fluid, a standard site, and some general standardized BAL procedures.

These trials are necessary for standardization of the BAL method in laboratory animals. The present study focused on standardizing 3 aspects of the BAL protocol. Firstly, we investigated the optimum number of times lung lavage should be performed.

Secondly, we determined which fraction of BAL fluid should be used to measure protein levels. Lastly, we determined whether the BAL fluid of the right or left lobes stood for the whole lungs.

Standardization of the BAL protocol will help reduce the variability between laboratories and improve the reproducibility and reliability of the BAL procedure. Rats were acclimated for at least 1 week. All experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with Association for Assessment and Accreditation of Laboratory Animal Care international guidelines.

Three individual experiments were performed. Here, we have used the term "suction frequency number" in BAL procedure to mean the administration and aspiration in each fraction with the same lavage fluid.

We have used the term "lavage fraction number" to mean the counted fraction number when we use fresh fluid buffer for each separate lavage. The same terms apply correspondingly to the following.

First, we compared the cell number and protein concentration of BAL fluid based on suction frequency number 1, 2, or 3 times with the same lavage fluid and lavage number up to 5. Second, 2 groups of rats were exposed to either bleomycin or saline by intratracheal instillation, and then performed BAL at 7 days after intratracheal instillation for cell counting and protein concentration measurement. Last, BAL was performed in the whole lungs or right lung lobes.

Five or six rats were used in each group and the experiment was repeated twice. Bronchoalveolar lavage based on suction times. Rats were anesthetized with isoflurane. The abdominal cavity was opened, exsanguination was performed via the aorta abdominalis, and the chest cavity was dissected to expose the lung and the trachea.

To identify the effects of suction frequency on contents of lavage fluid, 3 groups were separated by suction frequency: Lavage was performed 5 times in all groups and each aspirated BAL fluids was collected in a 15 ml conical tube, respectively. BAL cells were suspended in 1 ml of saline, stained with Turk's solution, and counted using a hemocytometer Neubauer, Marien-feld, Germany.

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Rats were divided into 2 groups: Each rat was anesthetized with isoflurane and intratracheal instillation was performed with saline or 2. After 1 week, we performed BAL with suction 2 times and lavage 5 times as described above. Determination of total protein and lactate dehydrogenase level from BAL fluid. Student's t-test was used to determine whether there was a statistically significant difference among the groups.

We observed a tendency for the number of lavaged cells to be lower with a single suction frequency than with 2 or 3 times of suction frequencies, but no significant differences were observed in the summation of the cell counts Fig.

Next, we evaluated the pattern of counted cells in each fraction of BAL fluid in the normal rats. The decrease in cell count took place over the sequence of lavage, and the first and the second lavage fraction contained similar numbers of cells Fig.

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