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. 2022 Jul;101(7):552-563.
doi: 10.1002/cyto.a.24558. Epub 2022 Apr 27.

Proliferative and anti-apoptotic fractions in maturing hematopoietic cell lineages and their role in homeostasis of normal bone marrow

Stefan G C Mestrum  1   2 , Roanalis B Y Vanblarcum  2 , Roosmarie J M Drent  2 , Bert T Boonen  3 , Wouter L W van Hemert  3 , Frans C S Ramaekers  1   4 , Anton H N Hopman  1 , Math P G Leers  2
Affiliations

Proliferative and anti-apoptotic fractions in maturing hematopoietic cell lineages and their role in homeostasis of normal bone marrow

Stefan G C Mestrum et al. Cytometry A. 2022 Jul.

Abstract

Recent developments in clinical flow cytometry allow the simultaneous assessment of proliferative and anti-apoptotic activity in the different hematopoietic cell lineages and during their maturation process. This can further advance the flow cytometric diagnosis of myeloid malignancies. In this study we established indicative reference values for the Ki-67 proliferation index and Bcl-2 anti-apoptotic index in blast cells, as well as maturing erythroid, myeloid, and monocytic cells from normal bone marrow (BM). Furthermore, the cell fractions co-expressing both proliferation and anti-apoptotic markers were quantified. Fifty BM aspirates from femoral heads of patients undergoing hip replacement were included in this study. Ten-color/twelve-parameter flow cytometry in combination with a software-based maturation tool was used for immunophenotypic analysis of Ki-67 and Bcl-2 positive fractions during the erythro-, myelo-, and monopoiesis. Indicative reference values for the Ki-67 and Bcl-2 positive fractions were established for different relevant hematopoietic cell populations in healthy BM. Ki-67 and Bcl-2 were equally expressed in the total CD34 positive blast cell compartment and 30% of Ki-67 positive blast cells also showed Bcl-2 positivity. The Ki-67 and Bcl-2 positive fractions were highest in the more immature erythroid, myeloid and monocytic cells. Both fractions then gradually declined during the subsequent maturation phases of these cell lineages. We present a novel application of an earlier developed assay that allows the simultaneous determination of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices in maturing hematopoietic cell populations of the BM. Their differential expression levels during the maturation process were in accordance with the demand and lifespan of these cell populations. The indicative reference values established in this study can act as a baseline for further cell biological and biomedical studies involving hematological malignancies.

Keywords: Bcl-2; Ki-67; anti-apoptosis; hematopoietic cell lineages; maturation; multiparameter flow cytometry; normal bone marrow; proliferation.

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Conflict of interest statement

F.C.S.R. is CSO and QA manager at Nordic‐MUbio, Susteren, The Netherlands.

Figures

FIGURE 1
FIGURE 1
Immunohistochemical staining pattern of normal bone marrow (BM) tissue from a femoral head with the Ki‐67 antibody MIB‐1 and Bcl‐2 antibody Bcl‐2/100 (10×, 40×, and 100× magnification). Cells positive for Ki‐67 or Bcl‐2 can be recognized by their brown staining. In normal BM, the majority of cells are positive for Ki‐67 and show a variable expression. Little cells are positive for Bcl‐2 in normal BM tissue and Bcl‐2 positivity is focused in specific regions. Ki‐67 positive cells were more abundant in the normal BM than Bcl‐2 positive cells.
FIGURE 2
FIGURE 2
Gating strategy of Ki‐67 and Bcl‐2 in the blast cells (A) and during the erythro‐ (B), myelo‐ (C) and monopoiesis (D) according to the threshold that was based on their respective IgG1 isotype controls. After gating the Ki‐67 or Bcl‐2 positive fraction, the maturation pathway for the total cell population and for the Ki‐67/Bcl‐2 positive fraction was drawn in the Infinicyt software. For erythroid cells, the CD117 versus HLADR plot was used to draw the maturation pathway. The maturation of the myeloid cells was drawn by the CD13 versus CD11b plot or the CD13 versus CD16 plot. The maturation of the monocytic cells was plotted by assessment of the CD64 versus CD14 expression. After determining the total number of cells and the Ki‐67/Bcl2 positive cells per maturation step of the erythro‐, myelo‐ and monopoiesis, the Ki‐67/Bcl‐2 positive fractions of cells per maturation step was calculated.
FIGURE 3
FIGURE 3
Gating procedure for the flow cytometric double staining of Ki‐67 and Bcl‐2 for (A) blast cells, (B) erythroid cells, (C) myeloid cells, and (D) monocytic cells. Gating thresholds were based on the IgG1 isotype control‐FITC versus IgG1 isotype control‐PE‐CF594 plot for each of the cell populations. After determining these thresholds, the Ki‐67 negative/Bcl‐2 negative, Ki‐67 positive/Bcl‐2 negative, Ki‐67 negative/Bcl‐2 positive, and Ki‐67 positive/Bcl‐2 positive fractions were determined for the different cell populations.
FIGURE 4
FIGURE 4
Graphic representation of the Ki‐67 positive and the Bcl‐2 positive fraction of blast, erythroid, myeloid, and monocytic cells. While the Ki‐67 positive fraction was highest in erythroid cells, a low Bcl‐2 positive fraction was observed. in myeloid cells, a low Ki‐67 and low Bcl‐2 positive fraction was seen. Monocytic cells displayed intermediate numbers of cells with expression of Bcl‐2 and low numbers with expression of Ki‐67. Blast cells showed an intermediate frequency of cells with expression of Ki‐67 and Bcl2.
FIGURE 5
FIGURE 5
Ki‐67 positive fractions and Bcl‐2 positive fractions in the different maturation stages of erythro‐, myelo‐ and monopoiesis. Black solid lines in each graph indicate the mean percentages of Ki‐67 positive or Bcl‐2 positive events for each maturation stage. The areas surrounding the solid line illustrate the standard deviation. For stage 0, the lineage‐committed blasts of the erythro‐, myelo‐ and monopoiesis were displayed. The Ki‐67 and Bcl‐2 positive fractions are highest in the early erythropoiesis (near 100%) and declined to 90%, 30% and 0%, respectively. During the early stages of the myelopoiesis, approximately 50% of the cells were positive for Ki‐67, while 50% of the cells were positive for Bcl‐2. Subsequently, the Ki‐67 positive fraction rose slightly to 60%, followed by a gradual decline to 0%. The Bcl‐2 positive fraction gradually declined to approximately 0% as the myeloid cells become mature. At stage 1 of the monopoiesis, the Ki‐67 and Bcl‐2 positive fractions were both 40%, and gradually declined to 0% as these cells undergo the maturation process. Morphological staging is indicated by capital letters, Arabic numbers and Roman numbers as follows. (A) early erythroid progenitors; (B) proerythroblasts; (C) basophilic erythroblasts; (D) polychromatophilic erythroblasts; (E) orthochromatophilic erythroblasts; 1: myeloblasts; 2: promyelocytes; 3: myelocytes; 4: metamyelocytes; 5: band cells; 6: neutrophils; I: monoblasts; II: promonocyte; III: monocytes.
FIGURE 6
FIGURE 6
Flow cytometric double immunostaining of blast cells, erythroid cells, myeloid cells and monocytic cells. (A) Graphical representation of double staining for Ki‐67 and Bcl‐2 in the different cell populations. Horizontal lines depict the mean fraction of cells, and the whiskers indicate the standard deviation. (B) Tabular visualization of double staining for Ki‐67 and Bcl‐2 in blast cells, erythroid cells, myeloid cells and monocytic cells. The blast cell population showed the largest fraction of Ki‐67/Bcl‐2 double positive cells of all myeloid cell compartments. In contrast, the committed cell populations (erythroid, myeloid, and monocytic cells) comprised almost no Ki‐67/Bcl‐2 double positive cells. Blast cells displayed an almost equal distribution of Ki‐67 and Bcl‐2 positivity. A large fraction of erythroid cells was Ki‐67 positive, while little cells were Bcl‐2 positive. The majority of myeloid cells were Ki‐67/Bcl‐2 double negative, while the other fractions were small. Monocytic cells displayed the largest Bcl‐2 positive fraction of all cell populations, while only a small number of cells were Ki‐67 positive.
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