Standardization of platelet‐derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?
Background: Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. Objectives: The objectives were (i) to standardize FCM settings for PMP counts on a routine instrum...
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| Published in | Journal of thrombosis and haemostasis Vol. 7; no. 1; pp. 190 - 197 |
|---|---|
| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Oxford, UK
Blackwell Publishing Ltd
01.01.2009
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1538-7933 1538-7836 1538-7836 |
| DOI | 10.1111/j.1538-7836.2008.03200.x |
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| Abstract | Background: Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. Objectives: The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size‐calibrated fluorescent beads; (ii) to determine intra‐instrument and interinstrument reproducibility; and (iii) to establish PMP values in healthy subjects. Methods: Using a blend of size‐calibrated fluorescent beads (0.5 and 0.9 μm) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra‐instrument and inter‐instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet‐free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers. Results: This calibrated‐bead strategy allowed full long‐term control of the FCM‐based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 μL−1 (1014–3039 μL−1) vs. 656 μL−1 (407–962 μL−1)]. Conclusions: The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies. |
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| AbstractList | Background: Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. Objectives: The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size‐calibrated fluorescent beads; (ii) to determine intra‐instrument and interinstrument reproducibility; and (iii) to establish PMP values in healthy subjects. Methods: Using a blend of size‐calibrated fluorescent beads (0.5 and 0.9 μm) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra‐instrument and inter‐instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet‐free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers. Results: This calibrated‐bead strategy allowed full long‐term control of the FCM‐based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 μL−1 (1014–3039 μL−1) vs. 656 μL−1 (407–962 μL−1)]. Conclusions: The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies. Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size-calibrated fluorescent beads; (ii) to determine intra-instrument and inter-instrument reproducibility; and (iii) to establish PMP values in healthy subjects. Using a blend of size-calibrated fluorescent beads (0.5 and 0.9 mum) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra-instrument and inter-instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet-free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers. This calibrated-bead strategy allowed full long-term control of the FCM-based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 microL(-1) (1014-3039 microL(-1)) vs. 656 microL(-1) (407-962 microL(-1))]. The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies. Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized.BACKGROUNDPlatelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized.The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size-calibrated fluorescent beads; (ii) to determine intra-instrument and inter-instrument reproducibility; and (iii) to establish PMP values in healthy subjects.OBJECTIVESThe objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size-calibrated fluorescent beads; (ii) to determine intra-instrument and inter-instrument reproducibility; and (iii) to establish PMP values in healthy subjects.Using a blend of size-calibrated fluorescent beads (0.5 and 0.9 mum) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra-instrument and inter-instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet-free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers.METHODSUsing a blend of size-calibrated fluorescent beads (0.5 and 0.9 mum) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra-instrument and inter-instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet-free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers.This calibrated-bead strategy allowed full long-term control of the FCM-based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 microL(-1) (1014-3039 microL(-1)) vs. 656 microL(-1) (407-962 microL(-1))].RESULTSThis calibrated-bead strategy allowed full long-term control of the FCM-based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 microL(-1) (1014-3039 microL(-1)) vs. 656 microL(-1) (407-962 microL(-1))].The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.CONCLUSIONSThe present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies. |
| Author | ROBERT, S. DIGNAT‐GEORGE, F. PONCELET, P. LACROIX, R. ARNAUD, L. SAMPOL, J. GIRAUDO, L. HAUCHARD, A. |
| Author_xml | – sequence: 1 givenname: S. surname: ROBERT fullname: ROBERT, S. – sequence: 2 givenname: P. surname: PONCELET fullname: PONCELET, P. – sequence: 3 givenname: R. surname: LACROIX fullname: LACROIX, R. – sequence: 4 givenname: L. surname: ARNAUD fullname: ARNAUD, L. – sequence: 5 givenname: L. surname: GIRAUDO fullname: GIRAUDO, L. – sequence: 6 givenname: A. surname: HAUCHARD fullname: HAUCHARD, A. – sequence: 7 givenname: J. surname: SAMPOL fullname: SAMPOL, J. – sequence: 8 givenname: F. surname: DIGNAT‐GEORGE fullname: DIGNAT‐GEORGE, F. |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/18983485$$D View this record in MEDLINE/PubMed |
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| Snippet | Background: Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow... Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry... |
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| SubjectTerms | beads Blood Platelets Calibration Cell-Derived Microparticles - pathology flow cytometry Flow Cytometry - methods Flow Cytometry - standards Humans megamix microparticles Microspheres Observer Variation Particle Size Reference Standards standardization |
| Title | Standardization of platelet‐derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies? |
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