Consistent quantitative gene product expression: #2. Antigen intensities on bone marrow cells are invariant between individuals

Abstract

Five reference populations in bone marrow specimens were identified by flow cytometry using specific combinations of reagents in order define the variation of gene product expression intensities both within and between individuals. Mature lymphocytes, uncommitted progenitor cells, promyelocytes, mature monocytes and mature neutrophils can be reproducibly identified as distinct clusters of events in heterogeneous, maturing bone marrow specimens. Support Vector Machines were used to identify the reference populations in order to reduce subjective bias in manually defining boundaries of these populations since they were not discretely separated from the remainder of the cells. Reference populations were identified in 50 randomly selected bone marrow aspirates obtained over a period spanning 3 years and 6 months from pediatric patients following chemotherapy for acute myeloid leukemia (AML). The quantitative expression of gene products (cell surface antigens) and light scattering characteristics on these stressed specimens were demonstrated to be tightly regulated both within individuals and between individuals. Within an individual most gene products (CD45, CD34, CD14, CD16, CD64, CD33) demonstrated limited variability with a standard deviation of <0.20 log units while CD13 and CD36 exhibited broader variation >0.25 log units. Surprisingly, with the exception of CD33, the variation of the mean intensities of each antigen between individuals was even less than the variation within an individual. These data confirm that the amounts of gene products expressed on normal developing cells are highly regulated but differ in intensities between different lineages and during the maturational pathway of those lineages. The amounts of gene products expressed at specific stages of development of each lineage are a biologic constant with minimal variation within or between individuals. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

Keywords: bone marrow aspirates; flow cytometry; quantitative antigen expression; support vector machines.

Figure 1

Identification of reference populations in stressed bone marrow specimens (all intensity units are in logarithmic scale; all frequency units are in linear scale). A, B: Mature lymphocytes were identified based on high expression of CD45 and low expression of log SSC (Purple). C, D: Uncommitted progenitor cells were identified by high expression of CD34 and intermediate expression of CD33 (Red). E, F: Promyelocytes expressed high levels of SSC (A) without expression of HLA‐DR or CD11b (Blue). G, H: Mature monocytes were defined based on high levels of CD14 and CD33 (Green). I, J: Mature neutrophils expressed high levels of SSC as well as CD16 and CD13 (Gold) (The reference population colors are maintained in subsequent figures).

Figure 2

CD45 intensity variation on reference populations for 50 pediatric bone marrow specimens. A: Variability of CD45 on lymphocytes (purple) and uncommitted progenitor cells (red). Each patient has 8 replicates for these two populations. The blue line represents the parameter mean averaged over all patients. Within patient variability is depicted by the whiskers (±1 SD). The average between patient variability (±1 SD) of the means is shown as the green lines. The histogram insert is added as a reference to visually demonstrate how little variation is observed for CD45 expression on lymphocytes (1 SD = 0.11 log units). B: CD45 intensity variability for promyelocytes (blue) and mature monocytes (green) assayed in a single reagent combination. The parameter means are shown as a blue line. Within patient variability (±1 SD) is demonstrated by the whiskers while the between patient variability (±1 SD) is depicted by the green lines.

Figure 3

Side scatter (SSC) variation for reference populations in 50 pediatric bone marrow specimens. A: Variability of SSC is displayed for lymphocytes (purple, with replicates) and monocytes (green). The parameter means are shown as a blue line. Within patient variability (±1 SD) is demonstrated by the whiskers while the between patient variability (±1 SD) is depicted by the green lines. B: Variability of uncommitted progenitor cells (red, with replicates) and promyelocytes (blue). The parameter means are shown as a blue line. Within patient variability (±1 SD) is demonstrated by the whiskers while the between patient variability (±1 SD) is depicted by the green lines.

Figure 4

CD45/log SSC relationships for reference populations for 50 pediatric bone marrow specimens. A: The CD45 vs. SSC position of mature lymphocytes (purple), uncommitted progenitor cells (red), mature monocytes (green), promyelocytes (blue), mature neutrophils (gold). The dashed ovals represent the between patient variability of CD45 (± 2 SD, y‐axis) and SSC (± 2 SD, x‐axis). B: Normalization of the position of the reference populations to lymphocytes. The lymphocytes (purple) were moved to the same location and the corresponding positions of the other reference parameters were also translocated using the same vector for each patient. The solid ovals represent the variation of the normalized positions in the CD45 dimension (±2 SD, y‐axis) and the SSC dimension (±2 SD, x‐axis).

Figure 5

Variation of intensities of CD34, CD14, and CD33 on 50 pediatric bone marrow specimens. A: CD34 variation on uncommitted progenitor cells showing 8 replicates, within patient variation (whiskers), between patient variation (green lines) and parameter mean (blue line). The proportion of uncommitted progenitor cells is variable (black lower line). Frequency is depicted on the y‐axis on the right side of the plot. B: CD14 expressed on mature monocytes showing the within patient variation (whiskers), between patient variation (green line) and parameter mean (blue line). C: CD33 expression on mature monocytes shows tight within patient variation (whiskers) but broader between patient variation (green line).