Aberrant Ki‐67 expression in normal bone marrow revealed by multiparameter flow cytometric analysis

Bockstaele, Dirk R. Van; Lan, Jun-Cai; Snoeck, Hans-Willem; Korthout, Marcel; Bock, Rudolf; Peetermans, M.

doi:10.1002/cyto.990120108

Cited by 26 publications

(10 citation statements)

References 57 publications

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“…The results in the present study are in concordance with results found in earlier studies (3,4,22), in which BM samples were analyzed for Ki-67 expression by one-or two-parameter flow cytometry or by immunohistochemistry. Noteworthy, Van Bockstaele et al could not detect any convincing Ki-67 positivity on nuclei from normal BM samples (5). They stated that in normal bone marrow the cycling compartment consist Table 2.…”

Section: Discussionmentioning

confidence: 98%

“…Despite the widespread use of Ki-67 in histopathology, its use in flow cytometric analysis of hematologic malignancies is sparse (see for example (3)(4)(5)). A possible explanation is that since 1983, at the time of the first description of this monoclonal antibody by Gerdes et al (6), only one-, and 10 years later two-fluorescence parameter flow cytometry was available.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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“…Moreover, some reports suggest that aberrant patterns of Ki-67 expression are demonstrated by benign cell populations, including drug-treated lymphocytes 36 and normal hematolymphoid elements. 37 In the present study, we investigated the relationship between lymphoma grade and the FCI analysis of FSC and CD71 expression, and we propose the latter as a surrogate marker for proliferative activity. Because our original study was designed to assess our ability to grade FLs, we only studied these FCI parameters in CD10+ mature B-cell malignant neoplasms.…”

Section: Discussionmentioning

confidence: 99%

The Usefulness of CD71 Expression by Flow Cytometry for Differentiating Indolent From Aggressive CD10+ B-Cell Lymphomas

Borowitz

Weir

2006

Am J Clin Pathol

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We studied 34 low- and 30 high-grade CD10+ B-cell lymphomas. Forward light scatter (FSC) and CD71 fluorescence intensity (CD71i) of tumor cells were measured and normalized by corresponding values for resting T cells. Significant differences in CD71i values between low- and high-grade lymphomas were observed by the Mann-Whitney U test (P < .001) and receiver operating characteristic (ROC) curve analysis (P < .001). FSC was not significantly different between low- and high-grade lymphomas; the area under the ROC curve was less than that for CD71i. Neither FSC nor CD71i significantly differentiated follicular lymphoma (FL) grades. A comparison of all FLs (grades 1-3) and non-FL high-grade lymphomas (Burkitt lymphoma [BL] and large B-cell lymphoma [LBCL]) showed significant differences in CD71i (P < .001) and FSC (P = .021). Differences were significant in CD71i and FSC between FL and LBCL (P < .001) but not between FL and BL. CD71i is more potent than FSC for distinguishing CD10+ low- from high-grade lymphomas and FL from non-FL high-grade lymphomas. Sensitivity and specificity are limited owing to inability to identify FL3. In ROC analysis, a high value for CD71i can identify BL and LBCL with a high degree of certainty.

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“…These proliferation markers include the presence of surface HLA class I1 antigens, interleukin-2 receptors (CD25), and transferrin receptors (CD74) (7); the presence of intracellular proliferation-related nuclear antigens such as Proliferating Cell (3) and is a prognostic marker of lymphomas (4,6), the amount of Ki-67 antigen is different in each cell type, and it is difficult to separate positively and negatively stained cells clearly (9). Furthermore, in a recent report, the utility of this marker in studying cell cycle of normal, non-stressed hematopoietic cells has been questioned (17). While PCNA is positive in GI and S-phase cells in cell cycle (12), the amount of PCNA changes according to cell cycle phase.…”

Section: Discussionmentioning

confidence: 99%

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S‐phase fraction, and surface phenotype using single laser instrumentation

1992

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We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (> 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.Key terms: Three-color fluorescence, flow cytometry, cell surface phenotype, cell cycle analysis, BrdUrd incorporation, 7-amino-actinomycin D Flow cytometry is routinely employed for immunophenotyping, measuring cell cycle, and determining DNA ploidy distribution. The simultaneous identification of surface phenotype and cell cycle has made it possible to analyze the proliferative status of specific cell subsets in heterogeneous suspensions even when the cells of interest occur at a low frequency.The most commonly employed DNA stain, propidium iodide (PI), has been used only with fluorescein isothiocyanate (F1TC)-conjugated monoclonal antibodies to simultaneously determine DNA ploidy and surface phenotype with a single argon laser (1,13). Due to the broad emission spectrum of PI, the use of phycoerythrin (PE)-conjugated antibodies has not been possible. In contrast, 7-amino-actinomycin D (7AAD), a DNA stain with a longer wavelength emission spectrum (> 650 nm), can be used in combination with FITC-conjugated as well as PE-conjugated antibodies (14).The cell cycle can be defined by two parameters: measurement of actual DNA synthesis (S-phase), and quantitation of total DNA and chromosome formation. Both PI and 7AAD can be used t o measure S-phase fraction. However, these methods rely on sophisticated mathematical models for determination of the percentage of cells in S-phase. In contrast, the detection of bromodeoxyuridine (BrdUrd) incorporation into DNA identifies cells actively synthesizing DNA during the period of BrdUrd exposure. This methodology yields the same information as 3H-thymidine uptake. Additional advantages of BrdUrd in studying cell cycle kinetics are that the BrdUrd can be administered to intact organisms as well as cells in culture, and can be administered at a time remote from when the cells are fixed and stained for analysis. When BrdUrd incorporation is combined with a DNA stain, it is possible to distinguish additional details of the cell cycle such as the proportion of qu...

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Aberrant Ki‐67 expression in normal bone marrow revealed by multiparameter flow cytometric analysis

Cited by 26 publications

References 57 publications

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

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

The Usefulness of CD71 Expression by Flow Cytometry for Differentiating Indolent From Aggressive CD10+ B-Cell Lymphomas

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S‐phase fraction, and surface phenotype using single laser instrumentation

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