Platelet measurement methods

The basic single-angle interval light-scattering method caimot accurately measure individual red blood cell or platelet volumes, but it can provide MCV and MPV. Red cells are bi-concave disks, and platelets ate rod to disk shaped Scattering intensities depend on the orientation in the flow cell. 

In principle, the two-angle interval method can produce all CBC parameters within a single measurement channel, uniquely providing ceU-by-ceU hemoglobin concentration. The mean of the concentrations provides an alternative (and direct) measurement of MCHC. The method also provides an alternative HGB measurement, because HGB may be set equal to (RBC x MCV x MCHC)/1000. This method, like the basic light-scattering method, uses the same flow cell to measure platelets and ted cells with the result that the method is capable of providing the CBC parameters RBC, HGB, HCT, MCV, MCHC, MCH, and PLT. The method can also count a sample s white blood cells if the sample s red blood cells have been lysed. 

Olas and Wachowicz (2002) investigated the effects of tranx-resveratrol and vitamin C on oxidative stress in blood platelets. The level of 02 in control blood platelets and platelets incubated with resveratrol or vitamin C was recorded using a chemiluminescence method. On the other hand, Oh and others (2006) reported the x02 quenching activities of various freshly squeezed fruit and vegetable juices by measuring their inhibitory effects on the rubrene oxidation induced by x02 from disproportionation of hydrogen peroxide by sodium molybdate in a microemulsion system. 

Again, the majority of these parameters are interrelated and highly dependent on the method used to determine them. Red blood cell count (RBC), platelet counts, and mean corpuscular volume (MCV) may be determined using a device such as a Coulter counter to take direct measurements, and the resulting data are usually stable for parametric methods. The hematocrit, however, may actually be a value calculated from the RBC and MCV values and, if so, is dependent on them. If the hematocrit is measured directly, instead of being calculated from the RBC and MCy it may be compared by parametric methods. 

X-ray diffraction techniques can be used to establish the structure of crystalline polymers. Measurements are typically made on crystalline lamellar platelets grown from dilute solution, fibres or stretched films. Such methods have been applied to several different inorganic polymers. For example, based on measurements on stretched samples of silicone rubber, poly(dimethylsiloxane) (Me2SiO) has been shown to possess a helical conformation (Figure 8.4). ... 

A benefit of FC over AVC/PTA or ELISA methods is the ability to measure more than one marker simultaneously. This can be exploited not only in the area of MP population selection but also in protocols concerned with the interaction of EMPs with other cells. In this regard, new studies exploring the interaction of EMPs with leukocytes and platelets have exploited one of FC s major advantages over other methods the ability to quantify MP cellular complexes [67, 93, 94], Similar methodologies have been employed with platelet microparticles (PMPs) or LMPs with great success and have contributed much of what we know about MPs functional aspects [34]. The approach of these assays is reasonably straightforward and may one day be used for clinical testing. 

The laboratory evaluation of platelet response to aspirin therapy has demonstrated response variability and nonresponsiveness, Based on different ex vivo methods, studies have shown wide variability in the prevalence of aspirin resistance (< I-54,7%) (45,49-58) (Table I). Potential reasons for these discrepancies include (/) wide variability in the criteria to define aspirin resistance, (//) variability in the methods to measure responsiveness, (///) the timing of the laboratory test after aspirin treatment, (/V) the duration of aspirin treatment, and (v) the dose of aspirin administered,... 

Flow cytometric determinations of P-selectin and activated GPIIb/llla receptor expression following ADP stimulation have been used to assess platelet inhibition by clopidogrel. Flow cytometry is also a cumbersome method and requires sophisticated instrumentation and well-trained technicians. The phosphorylation state of vasodilator-stimulated phos-phoprotein (VASP) is a specific intracellular marker of clopidogrel-induced P2Y 2 receptor inhibition and can also be measured by flow cytometry. Permeation of the membrane and the use of monoclonal antibodies specific for phosphory-lated VASP are required in this method (15,88). 

Reports of high incidences of aspirin resistance may be due to laboratory measurements based on nonspecific methods that do not isolate the response of platelet COX-1 to aspirin or due to an inadequate dose required to fully inhibit the COX-1 in selected patients. Clopidogrel nonresponsiveness is a consistent phenomenon observed in research studies conducted at multiple medical centers around the world. Data from small studies support that patients with high ex vivo platelet reactivity to ADP during and after percutaneous intervention may be at greatest risk for subsequent ischemic events. 

Fig. 11(a) shows the AFM image of an 11-layer mixed-stack CT film of octadecyl-TCNQ and (Me)2P scanned at room temperature with a scan area of 2x2 pm2 [29]. It can be seen from the image that the CT film consists of platelet microcrystal domains of a few micrometers in size in which a multi-layered structure with many steps is observed. An analysis of the cross-sectional profile revealed that the layered platelet microcrystal domains have a step of 3.3 nm thickness [29]. This is in good agreement with the d value measured by the X-ray diffraction method [28]. Therefore, it seemed that the X-ray diffraction peaks originate from the multi-layered structure inside the domains. Each layer in the domains apparently consists of biomolecular layers of octadecyl-TCNQ and (Me)2P because the layer thickness of 3.3 nm is larger than the molecular length (3.0nm) of octadecyl-TCNQ. The biomolecular layer structure also supports that the CT film is in a mixed-stack pattern. 

FIG. 10 Pressure curves for Na+-mica surfaces in a 2 mol/L NaCl solution calculated from Eq. (27) and converted for flat surfaces. Also represented is the swelling pressure for a Na-montmorillonite platelet system. Inset pressure curves measured using the direct force method [51]. 

The assay, introduced by Bom (1962a,b), has become a standard method in clinical diagnosis of platelet function disorders and of aspirin intake. Furthermore, the method is used in the discovery of antiplatelet drugs with the advantage of rapid measurement of a functional parameter in intact human platelets. However, processing of platelets during the preparion of PRP, washed or filtered platelets from whole blood results in platelet activation and separation of large-size platelets. 

A new highly sensitive method to study platelet aggregation based on the measurement of mean radius or particle size makes it possible to record kinetics of formation of micro- and macro-aggregates in real time... 

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