2,7-Fluorenediamine and 2,5-fluorenediamine as peroxidase reagents for blood smears

Our new histochemical methods for peroxidase activity, which do not require benzidine as a hydrogen donor, have been modified for hematological use. Diamine derivatives of fluorene (2,7-fluorenediamine and 2,5-fluorenediamine) were employed in place of benzidine and the result obtained was satisfactory. Two staining techniques were developed. (1) Smears of peripheral blood or bone marrow aspirates were fixed in 2.5 per cent glutaraldehyde solution for 1 minute. Smears were then washed in tap water and covered either by a saturated solution of 2,7-bluorenediamine or by a 0.05 per cent solution of 2,5-fluorenediamine in the presence of hydrogen peroxide for 5 minutes. After being washed in running tap water they were counterstained with Carazzi's hematoxylin solution for 10 minutes. (2) The second technique employs pretreatment of blood smears with 5 per cent CuSO4 solution and aldehyde fixation was omitted. Smears were counterstained by Giemsa stain. The nuclear chromatin structures were well preserved and nucleoli were easily distinguished in the immature cells. In the first method, clear brown granules were recognized in the cytoplasms. In the second method, peroxidase-positive granules were stained black and the staining of nucleus and cytoplasm resembled that of McJunkin's method.