Abstract Number: PB2005
Meeting: ISTH 2020 Congress
Theme: Vascular Biology » Inflammation and Sepsis
Background: Neutrophil Extracellular Traps (NETs) are composed of extracellular decondensed chromatin networks that play an important role in immunity. Simple and reliable identification of NETs has been challenging. Automated hematology analyzers such as the CellaVision® classify cell-derived remnants as smudge cells. We hypothesize that, in addition to the typical degenerated lymphocytes (DLs) forming smudge cells, a proportion of smudge cells as identified by the CellaVision® are actually NETs.
Aims: To study the correlation of high NET percent on blood smears with infection.
Methods: To test our hypothesis, we used morphologic characteristics, immunohistochemistry, immunofluorescence and flow cytometry to differentiate NETs from DLs. Smears with >20% smudge cells were classified morphologically as NETs vs. DLs; compared to control group, < 5% smudge cells. Medical chart review performed by blinded investigators, included patient demographics, presence of bacterial or viral infection occurring < 1 week of sample collection, and other comorbidities. Statistical analyses included two-sided t-test and chi-square.
Results: Within the study group of >20% smudge cells, 88 were morphologically designated as NETs, 8 as DL vs. 59 as control group. Smears from >20% smudge cells (NET-subclassification) stained strongly with myeloperoxidase (MPO), neutrophil elastase, leukocyte alkaline phosphatase, whereas DLs were negative. On Wright-Giemsa stain, cell remnants that were morphologically classified as NETs also stained for Sytox-Green. On flow cytometry, NETs are large and display extracellular DNA and MPO
[Figure 1: Characterization of Neutrophil Extracellular Traps and Degenerating lymphocytes. ]
. Comparing patients with >20% smudge and NET sub-classification to < 5% smudge cells, the formers had higher incidence of bacterial and viral infections (p=0.009/0.005 and p=0.009/0.008),
< 5% Smudge Cells n=59 | > 20% Smudge Cells n=96 NETs n=88 | > 20% Smudge Cells n=96 DLs n=8 | T-test < 5% vs. > 20% Smudge Cells | T-test < 5% vs. > NETs | T-test < 5% vs. DLs | T-test NETs vs. DLs | |
Bacterial Infection, n (%) | 11 (18.6%) | 35 (39.8%) | 1 (12.5%) | 0.0091 | 0.0045 | 0.66 | 0.073 |
Viral Infection (includes HIV, HBV, HCV), n (%) | 8 (13.6%) | 29 (33.0%) | 2 (25.0%) | 0.0085 | 0.0081 | 0.53 | 0.663 |
Lymphoproliferative Disorder, n (%) | 12 (20.3%) | 6 (6.81%) | 5 (62.5%) | 0.16 | 0.17 | 0.17 | 0.019 |
Myeloproliferative/ MDS, n (%) | 9 (15.3%) | 5 (5.7%) | 2 (25.0%) | 0.15 | 0.076 | 0.58 | 0.28 |
VTE/ATE, n (%) | 5 (8.5%) | 10(11.4%) | 1 (12.5%) | 0.544 | 0.56 | 0.76 | 0.93 |
WBC (x109/L), mean (SD) | 7.4 (4.8) | 8.5 (5.75) | 84.05 (69.6) | 0.014 | 0.18 | 0.017 | 0.018 |
Neutrophil (x109/L), mean (SD) | 5.24 (4.09) | 4.50 (4.0) | 5.35 (5.5) | 0.33 | 0.28 | 0.959 | 0.68 |
Age, median yrs (IQR) | 61 (68.1-43.5) | 48.0 (63.1-7.3) | 69.6 (80-65.2) | ||||
%Female | 42.4% | 44.3% | 50% |
[Table 1. Demographics and Comorbidity analysis for <5% Smudge Cells and >20% Smudge Cells, sub-classified as NET vs. DL(>80% cells in that category)]
.
Conclusions: This study is the first to identify NETs on peripheral smear evaluations as performed by a routine Hematology Autoanalyzer and to differentiate them from DLs using a reliable set of morphologic characteristics, immunohistochemical stains, and flow cytometry probes. This study also supports data that associate NETs with infections even in the absence of leukocytosis.
To cite this abstract in AMA style:
Reyes-Gil M, Yin D, Fedorov K, Barouqa M, Ikemura K, Kushnir M, Billett H. Neutrophilic Extracellular Traps (NETs) Are a Subset of Smudge Cells Identifiable by Peripheral Smear Autoanalyzers [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/neutrophilic-extracellular-traps-nets-are-a-subset-of-smudge-cells-identifiable-by-peripheral-smear-autoanalyzers/. Accessed March 21, 2024.« Back to ISTH 2020 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/neutrophilic-extracellular-traps-nets-are-a-subset-of-smudge-cells-identifiable-by-peripheral-smear-autoanalyzers/