Bio-maleimide as a generic stain for detection and quantitation of microparticles
A K Enjeti1, L Lincz, M Seldon
1Hunter Haematology research group, Mater Misericordiae Hospital, Newcastle, NSW, Australia. anoop.enjeti@mater.health.nsw.gov.au
Abstract:
Microparticles (MP) are small fragments of cytoplasm shed from a cell surface and their role in the pathophysiology of disease is being extensively investigated. A novel staining technique for quantifying total MP in peripheral blood was evaluated in this study. Evaluation of Bodipy-maleimide (or bio-maleimide) as a stain for quantifying total MP in peripheral blood by flow cytometry. Samples were obtained from 10 healthy donors after informed consent. Plasma was prepared by sequential centrifugation at 1500 g followed by 13,000 g and stained with Annexin V and bio-maleimide. Enumeration beads were added after 15 min of incubation with the stain and samples analyzed on a FACS Canto flow cytometer. Detection and quantification of MP by bio-maleimide staining was comparable with that by Annexin V. The total mean MP level with bio-maleimide staining was 34 +/- 19.7/microl (range of 11.6-68.1/microl) and with Annexin V staining it was 38.9 +/- 29.8/microl (range of 10.6 to 112.9/microl). There was no significant difference using a paired t-test and methods were comparable using a Bland-Altman plot. Bio-maleimide is a useful and inexpensive stain to measure total MP levels in peripheral blood by flow cytometry. This technique could be employed to study thrombotic risks in a variety of disease states.
Insights
Bio-maleimide is a cost-effective stain for quantifying microparticles (MP) in blood via flow cytometry. This method is comparable to Annexin V and can help assess thrombotic risks in various diseases.
Area of Science:
- Biochemistry
- Hematology
- Analytical Chemistry
Background:
- Microparticles (MPs) are cell fragments involved in disease pathophysiology.
- Accurate quantification of MPs in peripheral blood is crucial for research.
- Existing methods for MP quantification have limitations.
Purpose of the Study:
- To evaluate Bodipy-maleimide (bio-maleimide) as a novel stain for quantifying total MPs in peripheral blood.
- To compare the efficacy of bio-maleimide staining with Annexin V staining.
- To assess the utility of bio-maleimide for MP analysis in flow cytometry.
Main Methods:
- Peripheral blood samples from 10 healthy donors were analyzed.
- Plasma was isolated via sequential centrifugation.
- Samples were stained with Annexin V and bio-maleimide, followed by incubation.
- Enumeration beads were added, and samples were analyzed using FACS Canto flow cytometry.
Main Results:
- Bio-maleimide staining demonstrated comparable detection and quantification of MPs to Annexin V.
- Mean MP levels were similar between bio-maleimide (34 +/- 19.7/microl) and Annexin V (38.9 +/- 29.8/microl) staining.
- Paired t-test and Bland-Altman plots showed no significant difference, indicating method comparability.
Conclusions:
- Bio-maleimide is a valuable, inexpensive stain for measuring total MP levels in peripheral blood using flow cytometry.
- This technique offers a reliable alternative for MP quantification.
- The method can be applied to investigate thrombotic risks in diverse disease states.
