Table 2.

Techniques used to isolate and measure HDL-C subclasses.

Techniques	Advantages	Limitations
UC [51]	The first widely used method to separate the large buoyant HDL2 and the smaller, less buoyant HDL3 in plasma	The high salt concentration and the extreme g-force have been shown to significantly alter the composition and physicochemical properties of HDL that might influence some of the functional properties of the separated HDL fractions [59]
Single-step UTC: VAP assay [52]	Fast and use from whole plasma just one single predefined, narrow density ranges	Some HDL individual subpopulations cannot be isolated
Precipitation [53]	Separation based on ApoB depleted serum, cost, adequate access to this method in small laboratories	Proteins and apoE fraction confounders in HDL supernatant
Density gradient UTC [54]	Separation based on particle density, the standard method for lipoprotein method	High ionic strength and centrifugal force, High salt content that may affect the accuracy of the results
Gradient gel ND-PAGGE [55]	Separation by particle size, a sensitive method for evaluating insulin subclasses	Unable to separate pre$\beta$-2 populations, low access to laboratories, little information to predict cardiovascular disease
HPLC [50]	Separation based on the HDL particle size, rapid and accurate method	Access only in specialized laboratories, Albumin coelution with HDL fractions
NMR spectroscopy [56]	Separation based on the NMR signal of purified HDL, one of the convenient methods for measuring high volume samples, no prior sample manipulation	Lack of information on the composition of HDL subclasses, Inability to measure HDL subclasses with equal accuracy, Lack of detection of pre$\beta$-1HDL subclass
LCAT assay [57]	Separation based on the Fractional esterification rate, less cost, faster performance	May not measure the initial esterification rate and may not reflect the turnover of cholesterol
LipoPrint [58]	Clinically available measurement technique, lesser extent charge	Access in a small number of medical laboratories

LCAT, lecithin cholesterol acyltransferase; ND-PAGGE, non-denaturant polyacrylamide gradient gel electrophoresis; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; VAP, vertical auto profile; UC, density-gradient ultracentrifugation; UTC, ultracentrifugation.