Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses

Abstract

The use of flow cytometric enumeration of blasts in bone marrow aspirates has been of limited value in situations where blood contamination of the specimen is present. Assessment of sequential pulls of bone marrow aspirates from the same patient show decreasing proportions of blasts that are detected in later specimens. To address this problem, the intensity of CD16 on maturing neutrophils was compared for bone marrow biopsies, peripheral blood, and bone marrow aspirates. A comparison between bone marrow biopsy and aspirate specimens from the same individuals showed similar proportions of neutrophils with mature phenotype in most, but not all pairs. Other cell populations (total mature lymphocytes, monocytes, neutrophils, and blasts) were also similar between the two specimen types, with one exception of a patient with myelodysplasia, exhibiting a unique blast population in the biopsy that was not evident in the aspirate. The proportion of mature myeloid cells expressing a mature neutrophil phenotype (high levels of CD16) was found to be 17% (±6.7, n=47) in trephine marrow biopsy specimens. In contrast, marrow aspirates contained more of the mature neutrophil phenotype (38%±16, n=33) with about 1/3 of the aspirates indistinguishable from biopsies. Using a simple formula to normalize the aspirate specimens to the average neutrophil composition of marrow biopsies, it was possible to correct for the dilutional effect of added blood to both normal bone marrow aspirates and aspirates with elevated blast counts. These results suggest three alternative means of circumventing the problem of blood dilution of marrow aspirate specimens. (1) Blast counts by flow cytometry can be obtained from disaggregated biopsy specimens. (2) A bone marrow aspirate can be assessed for the proportion of mature neutrophils present and only those with low proportions (<30%) of phenotypic mature neutrophils are considered adequate for blast counting. (3) The aspirates with high proportions of mature neutrophils may be normalized based on the proportion of dim CD16 maturing myeloid cells to a level observed in bone marrow biopsies (based on an average mature neutrophil composition). Such an approach for identifying the amount of hemodilution in each specimen may enhance the utility of flow cytometry in enumeration of blasts in bone marrow, especially in cases where myeloblast count is crucial for prognosis, such as myelodysplastic syndromes (MDS).