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Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles
Y Yuana1, T H Oosterkamp, S Bahatyrova
1Department of Clinical Oncology, Leiden University Medical Centre, Leiden, the Netherlands.
Background:
Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio)vascular disorders and cancer. Most of these MPs are derived from platelets.
Objectives:
To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution.
Methods:
Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG1 isotype control was used as a negative control.
Results:
AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation=16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (dsph) ranged from 10 to 475 nm (mean: 67.5+/-26.5 nm) and from 5 to 204 nm (mean: 51.4+/-14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702x10(9) L(-1) plasma vs. 11-626x10(6) L(-1) plasma). After filtration of isolated MPs through a 0.22-microm filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean dsph: 37.2+/-11.6 nm), but not by flow cytometry.
Conclusions:
AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry.
Insights
Atomic force microscopy (AFM) offers a sensitive new method for detecting platelet-derived microparticles (MPs) in the nanosize range. This technique surpasses conventional flow cytometry for identifying these small vesicles in blood samples.
Area of Science:
- Biophysics
- Nanotechnology
- Hematology
Background:
- Microparticles (MPs) are cell-derived vesicles found in blood.
- Elevated MP levels are linked to cardiovascular disorders and cancer.
- Platelets are a major source of blood MPs.
Purpose of the Study:
- To evaluate atomic force microscopy (AFM) for detecting platelet-derived MPs.
- To determine the size distribution of these MPs using AFM.
Main Methods:
- Blood MPs were isolated and immobilized on antibody-coated mica.
- AFM in liquid-tapping mode detected CD41-positive MPs.
- Flow cytometry was used for parallel MP quantification.
Main Results:
- AFM measurements of CD41-positive MPs were reproducible.
- AFM detected MPs in the nanosize range (5-475 nm).
- AFM detected significantly more MPs than flow cytometry, especially after filtration.
Conclusions:
- AFM is a novel, sensitive method for detecting specific MP subsets.
- AFM can detect MPs in the nanosize range, below flow cytometry limits.
- This technique advances the study of MPs in disease.
