Background: The development of chronic lung disease is associated with an infiltration of activated immune cells which cause tissue damage by the release of oxygen radicals and enzymes. There has been controversy with most investigators suggesting only the presence of neutrophils contributes to the outcome(1). Most reported studies have used a standard differential stain to distinguish cells based on their morphology, where mature neutrophils are easily recognised but macrophages are not. Immunohistochemical stains (IHS) rely on cell-specific proteins to distinguish cell types. This should be more accurate and less observer dependent.

Samples: BAL was performed on infants ≤30wks gestation and the sample collected in a mucous trap. The cells were separated by centrifugation, counted and adjusted to 4×105cells/ml. 100μl of sample was added to a cytospin and spun onto slides, which were air dried and either differentially stained or stored at -20°C for later IHS.

Methods: The differential stain was a commercially available kit(Diff Quik, Dade). The IHS used the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique with monoclonal antibodies to the pan-macrophage marker CD68 (Dako) and the neutrophil-specific membrane antigens (Sigma). Counts were made on 300 cells per slide and the percentage of cell types present was compared using the Mantel-Haenszel χ2 test.

Results: Neutrophils counts were not significantly different between techniques (where n = 23, p=0.3) but there was a significant difference (n = 38, p<0.05) between macrophage counts, with the IHS counts being higher.

Conclusions: Routine differential staining may not be the best way of assessing macrophage numbers in BAL fluid as morphological differentiation is difficult. A technique based on the identification of cell-specific proteins by IHS is more discriminatory.