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Immunophenotyping of lymphocytes in bronchoalveolar lavage fluid. A new flow cytometric method vs standard

M Thomas1, M von Eiff, B Brandt

  • 1Department of Internal Medicine, Westfälische-Wilhelms Universität, Münster, Germany.

Chest
|August 1, 1995
PubMed

Insights

A new DNA-dye flow cytometry (FC) method reliably characterizes lymphocyte subsets in bronchoalveolar lavage fluid (BALF). This technique ensures accurate gating, offering comparable clinical reliability to the standard peroxidase anti-peroxidase (PAP) method for pulmonary disease diagnostics.

Area of Science:

  • Immunology
  • Pulmonary Medicine
  • Flow Cytometry

Background:

  • Accurate characterization of lymphocyte subsets in bronchoalveolar lavage fluid (BALF) is critical for diagnosing pulmonary diseases.
  • Traditional flow cytometry (FC) gating requires precise identification of lymphocyte populations, which can be challenging.
  • Assessing cell membrane integrity is a key factor in distinguishing viable lymphocytes from debris.

Purpose of the Study:

  • To introduce and evaluate a novel flow cytometry (FC) technique using a DNA-dye for assessing lymphocyte gate quality in BALF.
  • To compare the performance of this new FC method with the standard peroxidase anti-peroxidase (PAP) method for enumerating T lymphocyte subsets (CD3+, CD4+, CD8+).
  • To determine the reliability and clinical applicability of the DNA-dye FC method for BALF analysis.

Main Methods:

  • A prospective comparative study was conducted on BALF samples from 50 patients with various pulmonary conditions.
  • A novel FC technique was developed, incorporating a DNA-dye to evaluate cell membrane integrity and exclude non-DNA containing particles.
  • Lymphocyte subsets (CD3+, CD4+, CD8+) were quantified using both the new DNA-dye FC method and the standard PAP method after differential cell counts.

Main Results:

  • Both FC and PAP methods demonstrated excellent correlation for CD3 (r=0.81), CD4 (r=0.97), and CD8 (r=0.96) lymphocyte subsets (p < 0.05).
  • FC showed a tendency to slightly overestimate CD3+ cells compared to PAP (90.6% vs 85.8%).
  • There was good agreement between FC and PAP for CD4+ (45.0% vs 44.4%) and CD8+ (48.1% vs 46.7%) counts.

Conclusions:

  • The DNA-dye enhanced FC method provides a rapid and reliable approach for determining lymphocyte subsets in BALF.
  • The reliability of the new FC technique is equivalent to the standard PAP method in a clinical setting.
  • This novel FC method improves lymphocyte gating accuracy, aiding in the diagnosis and management of pulmonary diseases.

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Flow Cytometry01:23

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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