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Published on: April 3, 2014
Improved immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid (BALF) by flow cytometry
1Department of Clinical Laboratory, Peking Union Medical College Hospital, Beijing, China.
Insights
A new tri-color flow cytometric method accurately identifies lymphocytes in bronchoalveolar lavage fluid (BALF). This precise technique offers significant advantages over traditional methods for diagnosing lung diseases.
Area of Science:
- Immunology
- Pulmonary Medicine
- Diagnostic Cytometry
Background:
- Lymphocyte immunophenotyping in bronchoalveolar lavage fluid (BALF) is crucial for diagnosing interstitial lung disorders.
- Traditional peroxidase-anti-peroxidase (PAP) methods are time-consuming and experience-dependent.
- Flow cytometry (FCM) offers advantages but struggles with small cell counts and distinguishing lymphocytes from debris in BALF.
Purpose of the Study:
- To establish a precise and reliable tri-color flow cytometric (FCM) approach for lymphocyte phenotyping in BALF.
- To overcome limitations of existing methods for BALF analysis.
Main Methods:
- Developed a tri-color FCM assay using FITC-CD45/PE-CD14 antibodies for lymphocyte gating and propidium iodide (PI) for viability.
- Analyzed 43 BALF samples and peripheral blood controls using FCM and the conventional PAP method.
Main Results:
- The tri-color FCM method demonstrated significantly lower variation (CV<1.0%) compared to the PAP method (CV>9.8%).
- BALF T subset analysis showed greater clinical significance than peripheral blood.
- Distinct lymphocyte subset patterns were observed in sarcoidosis, idiopathic pulmonary fibrosis, lung cancer, and tuberculosis.
Conclusions:
- The tri-color FCM approach offers high precision and reliability for BALF lymphocyte phenotyping.
- This method is recommended as a routine test, particularly for BALF samples prone to contamination.
- The findings support the utility of FCM in diagnosing lung diseases based on BALF lymphocyte profiles.
Background:
The immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid (BALF) is of particular importance in the differential diagnosis of interstitial lung disorders. The standard method of lymphocyte phenotyping is peroxidase-anti-peroxidase technique (PAP). However, it was time-consuming and experience-dependent. Flow cytometric (FCM) analysis of BALF lymphocytes was introduced to overcome these disadvantages. Unfortunately, when the number of cells counted was small, FCM could not distinguish lymphocytes from other cells and particles in BALF by light scattering.
Methods:
We established a tri-color flow cytometric approach to phenotyping of lymphocytes in BALF. FITC-CD45/PE-CD14 antibodies were used to gate lymphocytes and exclude other contamination. Propidium iodide (PI) was introduced to distinguish lymphocytes from debris. Forty-three BALF species were tested by flow cytometer as well as peripheral blood as a control group by conventional PAP method.
Results:
The variation of FCM (CV<1.0%) was much lower that that of PAP method (CV>9.8%). Meanwhile, we found that BALF had more clinical significance than peripheral blood in T subset analysis (p<0.01). There were characteristic changes in some lung diseases. Both CD3 and CD4 were significantly increased with decreasing CD8 in sarcoidosis (n=14). Idiopathic pulmonary fibrosis (n=16) demonstrated the reverse tendency: CD8 rose but both CD3 and CD4 dropped. As for lung cancer (n=7), CD3 was normal but the CD4/CD8 ratio declined. Tuberculosis of the lungs (n=6) showed a normal CD3, CD4 and CD8.
Conclusions:
The high precision and reliability of tri-color flow cytometric approach to phenotyping of lymphocytes in BALF suggested that it should be used as a routine test, especially of BALF, which was often contaminated by inorganic particles.
