Assay accuracy is defined as the degree of closeness of achieved results relative to their actual (true) values. International standards exist for total IgG, IgA and IgM (CRM 470 and DA470k) . Therefore, these standards were used to assign values to HLC assay reference materials to ensure accurate results. An overview of the process of calibrator assignment for HLC assays is described below, using the IgA assays as an example.
9.4.1. Primary standards and internal reference standards
9.4.2. Kit calibrators and controls
HLC kit calibrators are prepared from pooled normal human sera, and kit controls from human sera containing high concentrations of polyclonal immunoglobulins. Values are assigned to kit calibrators using the internal reference materials (Figure 9.7). This is achieved by turbidimetry/nephelometry over five calibration curves, with the internal reference material assayed at four different dilutions with values across the curve range.
9.4.3. Calibration curves
The measuring ranges of HLC assays are dependent upon two factors: the slope of the respective calibration curve and the portion selected for the assay. The latter should be chosen to allow the maximum number of normal and abnormal clinical samples to be measured at the initial sample dilution.
|Specificity||Dilution||Approximate measuring range (g/L)|
|IgGκ||1/20||1.9 – 40.0|
|IgGλ||1/20||0.92 – 29.5|
|IgAκ||1/10||0.18 – 11.2|
|IgAλ||1/10||0.16 – 10.4|
|IgMκ||1/10||0.20 – 5.00|
|IgMλ||1/10||0.18 – 4.50|
Table 9.1. Measuring ranges for HLC assays on the Binding Site SPAPLUS.
Calibration curves are validated by the measurement of high and low control samples. It is also recommended that all laboratories take part in external quality assurance schemes, to allow comparison of performance and results among different test sites (Chapter 39).