Impact of phenotyping/flow cytometry technique on CD4/CD8 index examination in lung granulomatous diseases (LGD)

Background: There is no final consensus concerning normal value range of alveolar lymphocytes (AL), as a set point for auxiliary diagnostics in LGD. The important reason is a lack of AL typing and result analysis standardization. Aim: Evaluation what is an impact of typing principal technique, flourochrome conjugates with anti-CD4 and -CD8 antibodies (MoAbs), material hemolysis and cytometry gating for CD4/CD8 results. Methods: In bronchoalveolar lavage (BAL) obtained in 125 sarcoidosis, 65 hypersensitivity pnemonitis patients and 35 controls, CD4/CD8 index was gained with use of flow cytometry and standard CD4-FITC/CD8-PE MoAbs set, and compared with: a) CD4-PE/CD8-FITC set; b) CD4-APC/CD8-PE set (BD Multitest); c) CD4-FITC/CD8-PerCP; d) CD4-PerCP/CD8-FITC; e) anti-CD4 and -CD8 MoAbs in immunocytochemistry (ICC). We compared hemolysed and non-hemolysed samples and used cytometry gating techniques parallely: a) light side scatter/CD45 back-gating; b) CD3 gating; c) CD45 gating. Results: The results of CD4/CD8 index are similar for three result series in unhemolysed samples: a) CD4-FITC/CD8-PE set with CD45 gating; b) CD4-FITC/CD8-PE set with back-gating, if AL scatter gate was not contaminated; c) Multitest with CD45 gating. ICC produced declined CD4/CD8 index value (p Conclusions: We propose flow cytometry in non-hemolysed BAL samples with CD45 gating as standard method for CD4/CD8 examination in GLD. Hemolysis, ICC, CD3 gating and anti-CD4 MoAbs conjugated with PE either PerCP, should be omitted.