Methodological aspects of DNA image cytometry in normal bone marrow smears

The possibility of using DNA image cytometry in hematological material was evaluated in 27 Feulgen stained normal bone marrow smears. The 95% normal limits of DNA index (DI) determined separately for each cell population in bone marrow were 0.89-1.15, 0.93-1.09 and 0.92-1.12 for blasts, granulocytes and lymphocytes, respectively. The mean DI for blasts, granulocytes and lymphocytes was 1.02, 1.01 and 1.02, respectively. In the blasts population the mean percentage of cells in S+G2/M phase was 38.1% (S.D. 12.9%, range 14.3-65.6%), whereas the corresponding values for granulocytes and lymphocytes were below 1%. Calculation of DI and G0/G1 C.V. obtained in bone marrow smears subjected to Feulgen hydrolysis (5 N HCl, 22 degrees C) for different periods of time revealed the presence of a plateau of results between 60 and 120 min for all of the cell types. The intraobserver and interobserver reproducibility was confirmed by duplicate measurements of each subpopulation (P > 0.05). A comparison of DI and G0/G1 C.V. using smears from the same donors stained with the Feulgen method either straight away or after destaining from May-Grunwald-Giemsa confirmed that destained smears can be used, provided applying appropriately prepared reference cells. We conclude that image cytometry can be used reliably to analyze DNA content in hematological material.