Immunocytochemical distinction between primary and secondary granule formation in developing human neutrophils: Correlations with Romanowsky stains

Electron-microscopic studies with peroxidase cytochemistry have shown that primary (azurophilic) granules in human neutrophils are synthesized in promyelocytes, while secondary (specific) granules are formed in myelocytes. However, these studies were limited by the lack of specific markers for secondary granules and by the inability to make direct comparisons of electron-microscopic morphology with the light-microscopic appearance of the same cell. Thus secondary granules cannot be identified reliably and the relevance of these findings to the light-microscopic interpretation of clinical bone marrow specimens cannot be evaluated. To circumvent these problems, we developed a method to permit immunofluorescent demonstration of primary and secondary granule markers in cells stained with Romanowsky agents. Normal human marrow cells were stained with May-Grunwald-Giemsa and photographed. The slides were decolorized in buffered glycerine and saline for 24 hr and then stained with fluorescein- and rhodamine-conjugated monospecific antisera to human granulocyte myeloperoxidase, cathepsin G, elastase, lysozyme, and lactoferrin. The same cells were then located and examined for conjugate binding. Double stains with fluorescein- and rhodamine-labeled antisera offered the additional advantage of simultaneous identification of primary and secondary granules. This approach confirmed the partition of primary and secondary granule proteins and related their appearance to the maturation of developing neutrophils in normal human bone marrow.