**4.2 Quantitative assays**

Flow cytometry is the most widely employed quantitative technique. The gating of small particles continues to be a challenge but flow cytomtery continues to be the only robust technique which can demonstrate the cell of origin for the MP. This is an important asset of flow cytometry. However, there is significant variability amongst flow cytometers and the ISTH subcommittee on vascular biology recently conducted a workshop on standardization of MP by flow cytometry (Lacroix). It remains a popular approach for detection of MP for the following reasons:1)Rapid turn around time 2)Both fresh and frozen specimens may be used 3)The expression of two or more antigens on the MP may be simultaneously demonstrated 4)Easy method for quantification using commercial beads.

However it has the following drawbacks: 1) The detection of particles less than 0.3µm is difficult by flow cytometry as the detection is limited by particle size in the same order of magnitude wavelength of the laser ( about 488 nm 2) Different machines have different sensitivities 3) It is difficult to automate 4) Centrifugation speeds for sample processing are variable and not standardized (Freyssinet, 2005). Several new approaches to flow cytometry include using impedance flow cytometry and using Raman microspectrophotometry effect to cover the size and particle discrimination issues (Ayers, 2011).

The capture of MP into immobilized annexin V or cell specific antibodies using an ELISA based assay have ben the other major approaches (Enjeti, 2007). Solid phase assays have the advantage of picking up microparticles irrespective of size. However interference of soluble antigens, variable quality of antibodies used for antigen capture and non-exclusion of microsomes are some of the disadvantages.

#### **4.3 Nanoscale and newer technologies**

In the recent years there has been an adaptation of nanoscale technologies such as atomic force microscopy and nanoparticle measurement techniques. These methods claim to accurately measure particles in the nanoscale size range (Yuana). For example , one such nanoscale technique uses the brownian motion of these small particles to detect and measure them (Harrison, 2009). These methods are expensive, intensive to perform and not yet widely available (Lawrie, 2009). Moreover, the clinical utility of such techniques is not yet established. Recently a proteomic approach to analysis of MP has been described,

Microparticles: Role in Haemostasis and Venous Thromboembolism 13

Microparticles have therefore emerged as key role players in vascular biology and pathophysiology of thrombosis. They remain an important research tool and their clinical applications are being actively investigated with potential to be applied in diagnostic, prognostic and therapeutic arenas. They are small yet powerful effectors for the

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**6. References** 

2568.

however, the clinical utility of this approach is also as yet unkown (Howes ; Ramacciotti). Automated devices to analyse MP are also being developed (Wagner, 2010).

### **4.4 Measuring microparticles: Future directions**

There are several outstanding issues such as standardization of preanalytical and analytical variables as well as integration of the various approaches in measuring MP. Several novel approaches are now being considered. 'Megamix beads' is novel approach to standardizing of gating of microparticles using flow cytometry. It uses a mix of a 0.9um and 0.3um sized beads to try and capture all events within the gate set by the beads (Robert, 2009 ;Robert, 2011). One of the problems of using this approach is the lack of linearity in the relationship between the size of beads and forward sctatter at that particle size. A recent commercially available nanoscale technology known as 'Nanosight' has incorporated antibody tagging of small particles for accurate identification and counting in this size range (Harrison, 2009).
