Quantification of Cells Recovered by Bronchoalveolar Lavage: Comparison of Cytocentrifuge Preparations with the Filter Method

Willcox, Mary L.; Kervitsky, Alma; Watters, Leslie C.; King, Talmadge E.

doi:10.1164/ajrccm/138.1.74

Cited by 72 publications

(27 citation statements)

References 3 publications

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“…The number of cells (approximately 100 ϫ 10 6 to 150 ϫ 10 6 ), the cell type (approximately 80 to 90% monocytes/macrophages), and the volume of ELF (approximately 1 ml) are similar to the values that we and others have reported previously (1,2,4,7,10,26,29,33). Used with sensitive and specific drug assay techniques, this procedure permits an accurate estimation of the intrapulmonary drug concentrations in these compartments.…”

Section: Discussionsupporting

confidence: 80%

“…The number of cells from which drug was extracted was calculated by multiplying the number of cells per milliliter in BAL fluid times the volume (in milliliters) of BAL fluid that was centrifuged to produce the pellet. It has been noted, however, that centrifugation causes an average loss of 21% of the cells (33), so that the actual number of cells recovered postcentrifugation may be less than the number counted, and the actual antibiotic concentration may be proportionately more than we report here. The volume of ACs in the pellet suspension was calculated by using a mean macrophage cell volume of 2.42 l/10 6 cells (2).…”

Section: Methodscontrasting

confidence: 60%

See 1 more Smart Citation

Single-Dose Intrapulmonary Pharmacokinetics of Rifapentine in Normal Subjects

Conte

Golden²,

McQuitty

et al. 2000

Antimicrob Agents Chemother

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The intrapulmonary pharmacokinetics of rifapentine were studied in 30 volunteers who received a single, oral dose of rifapentine (600 mg). Subgroups of five subjects each underwent bronchoscopy and bronchoalveolar lavage (BAL) at timed intervals following drug administration. Drug concentrations, including the concentration of the primary metabolite 25-desacetyl rifapentine, were determined in plasma, BAL fluid, and alveolar cells (AC) by high-pressure liquid chromatography. The concentrations in epithelial lining fluid (ELF) were calculated by the urea diffusion method. The concentration-time data were fit to two-compartment (plasma) or one-compartment (AC and ELF) models. The peak concentrations in plasma, ELF, and AC, 26.2, 3.7, and 5.3 g/ml, respectively, occurred at 5, 5, and 7 h after drug administration, respectively. The half-lives and areas under the curve for plasma, ELF, and AC were 18.3 h and 520 g ⅐ h/ml, 20.8 h and 111 g ⅐ h/ml, and 13.0 h and 133 g ⅐ h/ml, respectively. Although the intrapulmonary rifapentine concentrations were less than the plasma rifapentine concentrations at all time periods, they remained above the proposed breakpoint for M. tuberculosis (0.5 g/ml) for the 48-h observation period. These data provide a pharmacokinetic rationale for extended-interval dosing. The optimum dosing regimen for rifapentine will have to be determined by controlled clinical trials.Rifapentine is an orally administered rifamycin derivative that has antituberculous activity and that is similar to rifampin (11,12,16,27). The MICs for sensitive strains are usually in the range of 0.03 to 0.12 mg/liter, and MICs for resistant strains are Ն8 mg/liter (15). Cross-resistance between rifapentine and rifampin is virtually complete (15). Cross-resistance between rifapentine and rifampin is virtually complete (15). Rifapentine has a longer elimination half-life than rifampin, allowing the possibility of less frequent (twice-or once-weekly) administration (17).There have been no previous reports of the intrapulmonary concentrations or pharmacokinetics of rifapentine in humans. The purpose of this study was to compare the concentrations of rifapentine in plasma, alveolar cells (ACs), and epithelial lining fluid (ELF) of normal volunteers and to compare the drug's pharmacokinetics in these three compartments. MATERIALS AND METHODSSubjects. The protocol was approved by the Human Research Committee of the University of California, San Francisco. Written informed consent was obtained from each subject by an experienced research nurse. Subjects were required to be 18 to 45 years of age. If the subjects were female, they were required to be nonlactating and not pregnant and to agree to use adequate contraception (e.g., barrier methods or abstinence) during the study and for 2 weeks following completion of the study. Women using oral contraceptives were required to agree to use a barrier method in addition for 1 month following the study. Subjects who were lactating or pregnant, had a history of intolerance to rifamycin dr...

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Section: Discussionsupporting

confidence: 80%

Section: Methodscontrasting

confidence: 60%

Single-Dose Intrapulmonary Pharmacokinetics of Rifapentine in Normal Subjects

Conte

Golden²,

McQuitty

et al. 2000

Antimicrob Agents Chemother

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“…The number of cells in 1.0 ml of pellet suspension was calculated to be equal to the number of cells in 1.0 ml of BAL fluid times 30. Because of cell loss during centrifugation, the actual number of cells recovered may be lower than the number counted and the antibiotic concentration may be approximately 20% higher than what we calculated (26). Differential cell counting was performed after spinning the specimen in a cytocentrifuge.…”

Section: Methodsmentioning

confidence: 94%

Intrapulmonary Pharmacokinetics of Linezolid

Conte

Golden

Kipps³

et al. 2002

Antimicrob Agents Chemother

284

185

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In this study, our objective was to determine the steady-state intrapulmonary concentrations and pharmacokinetic parameters of orally administered linezolid in healthy volunteers. Linezolid (600 mg every 12 h for a total of five doses) was administered orally to 25 healthy adult male subjects. Each subgroup contained five subjects, who underwent bronchoscopy and bronchoalveolar lavage (BAL) 4, 8, 12, 24, or 48 h after administration of the last dose. Blood was obtained for drug assay prior to administration of the first dose and fifth dose and at the completion of bronchoscopy and BAL. Standardized bronchoscopy was performed without systemic sedation. The volume of epithelial lining fluid (ELF) recovered was calculated by the urea dilution method, and the total number of alveolar cells (AC) was counted in a hemocytometer after cytocentrifugation. Linezolid was measured in plasma by a high-pressure liquid chromatography (HPLC) technique and in BAL specimens and AC by a combined HPLC-mass spectrometry technique. Areas under the concentration-time curves (AUCs) for linezolid in plasma, ELF, and AC were derived by noncompartmental analysis. Half-lives for linezolid in plasma, ELF, and AC were calculated from the elimination rate constants derived from a monoexponential fit of the means of the observed concentrations at each time point. Concentrations (means ؎ standard deviations) in plasma, ELF, and AC, respectively, were 7.3 ؎ 4.9, 64.3 ؎ 33.1, and 2.2 ؎ 0.6 g/ml at the 4-h BAL time point and 7.6 ؎ 1.7, 24.3 ؎ 13.3, and 1.4 ؎ 1.3 g/ml at the 12-h BAL time point. Linezolid concentrations in plasma, ELF, and AC declined monoexponentially, with half-lives of 6.9, 7.0, and 5.7 h, respectively. For a MIC of 4, the 12-h plasma AUC/MIC and maximum concentration/MIC ratios were 34.6 and 3.9, respectively, and the percentage of time the drug remained above the MIC for the 12-h dosing interval was 100%; the corresponding ratios in ELF were 120 and 16.1, respectively, and the percentage of time the drug remained above the MIC was 100%. The long plasma and intrapulmonary linezolid half-lives and the percentage of time spent above the MIC of 100% of the dosing interval provide a pharmacokinetic rationale for drug administration every 12 h and indicate that linezolid is likely to be an effective agent for the treatment of pulmonary infections.

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“…Other studies have not found that cytocentrifugation is responsible for loss of lymphocytes. In one study, it was found that differences in lymphocyte counts were due to the addition of serum to BALF [11]. In a second study, differences in lymphocyte counts were felt to be due to differences in methods used to fix and stain the cells [12].…”

Section: Discussionmentioning

confidence: 99%

“…Careful investigations have demonstrated that the method of processing of BALF can significantly affect data interpretation [7][8][9][10][11][12]. For example, the two most widely utilized methods, cytocentrifugation and membrane filtration, underestimate the content of lymphocytes and neutrophils, respectively [7,9,10].…”

Section: Technical Notementioning

confidence: 99%

Preparation of bronchoalveolar lavage fluid with microscope slide smears

et al. 1996

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The method of preparation of bronchoalveolar lavage fluid (BALF) for cytological examination can significantly affect the results of cellular quantitation. Investigations have shown that cytocentrifugation leads to an underestimation of the number of lymphocytes and membrane filter preparation to an underestimation of the number of neutrophils. As a simple alternative to these two techniques, BALF cells could be prepared by the microscope slide smear technique, which is familiar as the means for preparing peripheral blood for differential counts.In order to compare cell differentials determined by microscope slide technique with differentials resulting from cytocentrifugation, cells were isolated from 35 BALF samples using standard methods, and counted using a haematocytometer. Forty thousand cells in 200 µL were prepared by cytocentrifugation (3 min, 57×g; Cytospin 2) and 5×10 5 cells in 5 µL by microscope slide smear. Both samples were air-dried, stained using May-Grünwald Giemsa stain, and 600 cells were counted to obtain differentials. To test the adequacy of sampling by the microscope slide smear technique, known quantities of lymphocytes or neutrophils were added to fixed numbers of BALF cells, microscope slide smears prepared, and differentials determined on 600 cells. The resulting differentials were compared to the calculated differentials.Preparation Thus, microscope slide smear preparation is a simple and accurate method for the quantitation of bronchoalveolar lavage fluid cytology.

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Quantification of Cells Recovered by Bronchoalveolar Lavage: Comparison of Cytocentrifuge Preparations with the Filter Method

Cited by 72 publications

References 3 publications

Single-Dose Intrapulmonary Pharmacokinetics of Rifapentine in Normal Subjects

Single-Dose Intrapulmonary Pharmacokinetics of Rifapentine in Normal Subjects

Intrapulmonary Pharmacokinetics of Linezolid

Preparation of bronchoalveolar lavage fluid with microscope slide smears

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