Whole Blood Analysis of Leukocyte Platelet Aggregates

unit 6.15

Platelets play a critical role in the initial reaction to bleeding by rapidly forming a plug at sites of vascular injury. In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils (Michelson et al., 2002). The binding of platelets to monocytes and neutrophils is initiated primarily through platelet surface expression of P-selectin (CD62P) following activation-dependent degranulation (McEver, 2002). P-selectin in turn binds to PSGL-1 (P-selectin glycoprotein ligand-1, also known as CD162), which is constitutively expressed on the leukocyte surface. Leukocyte-platelet aggregates (LPAs) are therefore formed, and these are stabilized by several mechanisms, including the binding of leukocyte Mac-1 (CD11b/CD18, integrin aMp2) and platelet glycoprotein (GP) Iba (McEver, 2002).

Circulating monocyte-platelet aggregates have been demonstrated to be a more sensitive marker than platelet surface P-selectin for in vivo platelet activation (Michelson et al., 2001). Although the cellular interactions involved are complex, detection of LPAs by whole blood flow cytometry is relatively simple. While direct measurement of low-level platelet P-selectin expression is often difficult, even low-level P-selectin expression can initiate leukocyte binding. The presence of platelets associated with leukocytes is then easily detected using immunostaining for abundant platelet-specific markers. Flow cy-tometric analysis of LPAs involves gating on the desired leukocyte population through light scatter and at least one leukocyte-specific reagent such as CD14 to identify monocytes (bright) and neutrophils (dim). A second antibody that is platelet specific, typically CD41 (GPIIb), CD61 (GPIIIa), or CD42a (GPIX), will distinguish distinct platelet-positive and platelet-negative leukocyte subpopulations due to the high copy number of CD41, CD61, and CD42a on platelets. Platelets are quite heterogeneous in size (1 to 5 ^m in diameter) and platelet-derived microparticles (PDMP) also express P-selectin, which may explain the appearance of dim platelet-positive leukocytes whose fluorescence is just above that of the isotypic negative control antibody. LPAs of this type should not be overlooked, as circulating PDMP are often elevated in clinically relevant disease states (Michelson et al., 2002; Nieuwland and Sturk, 2002).

Review of unit 6.10 is strongly recommended, as many of the important considerations outlined therein are also critical in LPA analysis. For example, both choice of anticoagulant and blood sample handling can affect the measurement of LPAs. Expedient processing is also important due to the sensitivity of the assay and a high likelihood of in vitro platelet activation, resulting in an artifactually high number of LPAs.

0 0

Post a comment