Determination of the proliferative fractions in differentiating hematopoietic cell lineages of normal bone marrow

Abstract

Because of the proven prognostic value of Ki-67 as a proliferation marker in several types of solid cancers, our goal is to develop and validate a multiparameter flow cytometric assay for the determination of the Ki-67 expression in hemato-oncological diseases. The aim of the present study was to establish the reference values for the fraction of Ki-67 positive cells in and during maturation of individual hematopoietic cell lineages present in normal bone marrow. Aspirates derived from femoral heads of 50 patients undergoing a hip replacement were used for the flow cytometric quantification of Ki-67 expression in the different hematopoietic cell populations of healthy bone marrow. Furthermore, the proliferative index was investigated in detail for the maturation steps during erythro-, myelo-, and monopoiesis using recently described immunophenotypic profiles in combination with a software-based maturation tool. Reference values for the proliferative index were established for different relevant hematopoietic cell populations in healthy bone marrow. During maturation, the size of the Ki-67 positive fraction was the highest in the most immature compartment of the myeloid, monocytic, and erythroid cell lineages, followed by a steady decline upon cell maturation. While proerythroblasts showed a proliferative activity of almost 100%, the myelo- and monoblast showed a lower proliferative index of on average of 50%, indicating that a relatively large proportion of these cells exist in a quiescent state. In conclusion, we can state that when using a novel combination of immunophenotypic markers, the proliferation marker (Ki-67) and a software-based maturation tool, it was possible to determine the proliferative fractions in the diverse hematopoietic cell lineages in bone marrow, in particular during maturation. Using this approach, the proliferative indices for the normal myelo-, mono-, and erythropoiesis were determined, which can be used as a reference in future studies of hematologic malignancies originating from bone marrow. © 2018 International Society for Advancement of Cytometry.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…This allows others to analyze published data by alternative methods and better understand the published material. In the following manuscripts, you can find examples for MIFlowCyt checklists with different MIFlowCyt score values and original FCS data in the FlowRepository for Flow and mass cytometry . Since October 2018 MIFlowCyt compliance and reposition of original data are mandatory for Cytometry Part A publications .…”

Proliferative activity is disturbed in myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), and MDS/MPN diseases. Differences between MDS and MDS/MPN

“…Seventy‐eight patients diagnosed with MPN, MDS, or MDS/MPN (Table 1) and fifty individuals with non‐malignant hip lesions (also used in our earlier study) were included in this study (Nies et al, 2018). For only 26 of the 36 MDS patients and 6 of the 10 MDS/MPN patients, data for the erythropoiesis were available.…”

“…The antibody panels of the different tubes are shown in Table 2. Antibody characteristics can be found in our previous publication (Nies et al, 2018). Analysis of these tubes was performed using the Infinicyt v1.7 software package (Cytognos SL, Salamanca, Spain) as described previously (Nies et al, 2018).…”

“…Immunophenotypic analysis was performed using 10-color flow cytometry, as described previously (Nies et al, 2018). Four different antibody panels were used to determine the Ki-67 positive fraction during the erythro-, myelo-, and monopoiesis.…”

“…Our group has recently described and validated a multiparameter flow cytometric Ki‐67 assay for the objective determination of the proliferative activity in the diverse hematopoietic cell lineages in normal BM. Reference Ki‐67 values were established in the different hematopoietic cell lineages, while also its expression was quantified during the maturation process of the myelo‐, mono‐, and erythropoiesis (Supplemental Figure S1) (Nies et al, 2018).…”