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.

Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood

“…Future studies might choose to determine the purity of BM or to correct for peripheral blood contamination by methods that have been described previously. [45][46][47] A certain degree of peripheral blood contamination seems unavoidable given the hypocellularity of BM in the majority of RCC patients. Another limitation of our study and of most other studies evaluating the value of flow cytometry as a diagnostic tool in MDS, is that, although determining cell numbers by flow cytometry is fairly objective, scoring of dysplasia remains subjective.…”

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

“…Of note, several methods can be applied to evaluate the degree of hemodilution (e.g., the proportion of mature, CD16 þ neutrophils). 16,17 Within the current WHO proposal, 7 the presence of circulating blasts is included in the classification. It is associated with a worse clinical outcome, that is, increased risk of leukemic transformation and post-transplant relapse.…”

Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes

“…Moreover, samples can be diluted by peripheral blood. 38 Another important issue is the terminology of what constitutes a blast: the definition of blasts by FCM (myeloid as well as lymphoid), inclusion or exclusion of granular blasts, and the resemblance of monocytes and blasts should be defined and/or corrected in a uniform manner. 3 Myeloblasts may + (in combination with a myeloid marker, negative for lymphoid markers) and denotes the earliest stage of the myeloblast.…”