Monocyte aggregates: A clue to an unsuspected monoclonal B‐cell lymphoproliferative disorder
Authors: Lynh Nguyen, Ronald Cleveland, Lawrence Kass
Journal: American Journal of Hematology (2016)

To the Editor: In our daily pathologist's review of Wright-stained peripheral blood smears, we noted that a small number of patients with monocytosis showed monocyte aggregation, while other patients with monocytosis did not. We further investigated this population of patients to elucidate the significance of these monocyte aggregates and identify any similarities these patients might share. The Institutional Review Board at MetroHealth Medical Center at Case Western Reserve University approved this study. Leftover peripheral venous blood samples were obtained from patients during the course of routine care. Blood samples identified with monocytosis (>20% monocytes, absolute monocytosis 2.0 or greater) by the Sysmex Hematology Analyzer XE5000 were stained using Wright's stain and examined. α-Naphthyl Butyrate Esterase Activity (ANBE): Fluoride-sensitive nonspecific esterase activity, which uses α-naphthyl butyrate as substrate, is a property of monocytes. It is not present on the cell surfaces of other cell lineages. However, lymphocytes may show some punctate cytoplasmic α-naphthyl butyrate esterase activity (ANBE) reactivity with no notable membranous reactivity. Cytochemical staining for ANBE of the peripheral blood smears was performed as described previously 1. Flow Cytometry: The peripheral venous blood from four patients were processed and evaluated using a six-color flow cytometer (FACSCanto II, Becton Dickinson) after labeling with monoclonal antibodies against CD38, CD3, CD5, CD19, CD20, CD23, CD11b, CD15, CD13, CD33, CD14, and CD45 (Becton Dickinson, San Jose, CA). Erythrocytes were then lysed by adding FACS lysing solution (Becton Dickinson). The leukocytes were centrifuged, washed twice in phosphate-buffered saline (pH 7.4), and finally resuspended in 0.5% paraformaldehyde for analysis. The percentage of positive cells for each marker was determined using a flow cytometer in conventional manufacturer-provided configuration (FACSCanto II, Becton Dickinson). Electron Microscopy: Peripheral venous blood samples were obtained in a collection tube containing EDTA and centrifuged at room temperature at 400g for 15 min. For the study, 2 mL of whole blood was used from each patient. After removing the plasma, an equal volume of 2.5% phosphate-buffered glutaraldehyde was added without disturbing the buffy coat and incubated for 30 min at room temperature. The clotted buffy coat was removed using wood applicator sticks, and then kept in a vial of fresh 0.5% phosphate-buffered glutaraldehyde for 90 min at room temperature. Samples were incubated overnight in acidified uranyl acetate, fixed, and dehydrated. Sections were stained with acidified uranyl acetate followed by a modification of Sato's triple lead stain 2. These sections were examined in a FEI Tecnai Spirit (T12) transmission electron microscope with a Gatan US4000 4kx4k CCD. Over the course of three months, approximately 150–180 blood samples were reviewed for monocytosis. Unexpectedly, a subset of 13 patients showed monocyte aggregates (Supporting Information material). The aggregates consisted of two or sometimes three monocytes (Fig. 1A,B). At sites of monocyte adhesion, a concentration of azurophillic granules was noted (Fig. 1A,B). Monocytes have been described previously to aggregate to platelets in certain conditions 3, 4. However, in our study the monocytes adhered only to other monocytes and not to other cells. Electron micrographs of the monocyte aggregates demonstrated a concentration of lysosomes at the junction site. Also there were several cases where the cytoplasm of one monocyte seemed to enter the cytoplasm of the adjacent monocyte (Fig. 1C,D). Fluoride-sensitive nonspecific esterase stains and co-expression of CD13 and CD14 on flow cytometry confirmed that the aggregates consisted of monocytes. The aggregates displayed identical marker characteristics as the monocyte singlet fraction and could be distinguished as a separate cluster of cells with brighter expression of CD45 fluorescence and higher side scatter expression compared to the singlet fraction (Fig. 1E). Flow cytometric analysis was performed on a total of four patients, three of which were over the age of 80 years old. These patients did not have any known overlapping past medical histories. Interestingly in two of these three patients, flow cytometry revealed a monoclonal B-cell population not detected morphologically on peripheral blood smear (Fig. 1E). A third patient had a previous history of treated chronic lymphocytic leukemia (CLL) with no morphologic evidence of a persistent CLL at the time of this study. The one remaining patient with monocyte aggregates did not show a monoclonal B-cell population. A,B: Wright stain demonstrating concentration of azurophilic granules at the junction of two monocytes. C,D: Electron micrograph showing concentration of lysozymes at junction sites. In several cases, the cytoplasm of one monocyte seemed to flow into the cytoplasm of the adherent monocyte (arrows). E: Flow cytometry results showing concurrent monocyte aggregates encircled in blue and monoclonal B-cell population encircled in red. Coexistence of more than one hematologic disorder in the same individual suggests the versatility of expression of a multipotential stem cell. Monocyte aggregates could serve as a simple way of suspecting the simultaneous occurrence of another hematologic malignancy in an unrelated cell line (CLL lymphocytes). Why this occurs in some individuals with monocyte aggregates and not in others is as yet unknown. Large-scale studies of the parameters described were not possible in our pilot study due to extremely limited sample amounts. Mechanisms responsible for monocyte adhesion in our studies are not well understood and could be addressed in additional experiments if sample amounts permit. Recognition of monocyte aggregates could provide a quick and inexpensive morphologic clue to a coexisting B-cell lymphoproliferative disorder, and assist in identifying those samples, which require further analysis by flow cytometry. Also the significance of monocyte aggregates in patients who have untreated and treated CLL suggests an area for further study. Authors thank Joseph F. Tomashefski, M.D., Chairman of our Department of Pathology for his encouragement and support of this project. Lynh Nguyen, Ronald Cleveland, and Lawrence Kass* MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio Additional Supporting Information may be found in the online version of this article. Supporting Information Table 1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.