High-Sensitivity Flow Cytometry Provides Access to Standardized Measurement of Small-Size Microparticles—Brief Report

Abstract

Objective-Cellular microparticles (MP) are promising biomarkers in many pathological situations. Although flow cytometry (FCM) is widely used for their measurement, it has raised controversies because the smallest MP size falls below the detection limit of standard FCM (sd-FCM). Following recent technological improvements leading to high sensitivity FCM (hs-FCM), our objectives were (1) to evaluate the potential of hs-FCM for extended MP detection, (2) to set up a standardized protocol for MP enumeration, and (3) to compare MP counts obtained with both sensitivity levels. Methods and Results-Compared with sd-FCM, hs-FCM displayed improved forward scatter resolution and lower background noise, allowing us to discriminate previously undetectable small MP in plasma samples. Using fluorescent beads with appropriate sizes (0.1/0.3/0.5/0.9 m) and relative amounts, a new standardized hs-FCM MP protocol was set up and provided reproducible MP counts. Applied to coronary patient samples, it resulted into 8-to 20-fold increases in MP counts as compared with sd-FCM. Interestingly, the ratio between small and large MP varied according to clinical status but also depending on MP subset, suggesting access to new biological information. 3-5 Circulating MP are elevated in many prothrombotic and inflammatory disorders; cardiovascular, autoimmune, and infectious diseases; and cancer. 4,[6][7][8][9][10] Although MP counts may provide useful diagnostic/prognostic information, assessment of their pathophysiological relevance is hampered by methodological concerns. Flow cytometry (FCM) is the most commonly used technique, 11 allowing both enumeration and characterization of MP cellular origin with high throughput. The forward scatter (FS) parameter is generally used to define the analysis gate for MP. Unfortunately, because of their limited FS sensitivity, standard flow cytometers measure only a small part of the MP population. Recently, high-sensitivity flow cytometers with significantly improved light scatter detection became available. As we first reported, these instruments resolve a previously undetectable subpopulation of small-size MPs. Conclusion-Recent12 Among the technical improvements found in high sensitivity FCM (hs-FCM), a new option of the Beckman Coulter Gallios called W2 increases FS resolution by amplifying the FS signals collected at large angles. 13 Whether this option can be used in a standardized manner, to improve the detection of MPs derived from platelets (PMP), erythrocytes (Ery-MP), leukocytes (Leu-MP), and endothelial cells (EMP), remains an open question. Our objectives were then (1) to evaluate the impact of high-sensitivity flow cytometer technical improvement on the detection of small-size MP, (2) to set up a new standardized protocol based on calibrated microbeads for MP enumeration, and (3) to compare MP counts obtained at both sensitivity levels of FCM. MethodsBlood and plasma samples, MP preparation and flow cytometric analysis, size-conditioned MP filtration, intra-and inter-instrument reproducibi...

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