Kinetic Approach Extends the Analytical Measurement Range and Corrects Antigen Excess in Homogeneous Turbidimetric Immunoassays.

BACKGROUND: Homogeneous turbidimetric immunoassays are widely used in the clinical laboratory and offer short assay times, reduced reagent costs, and the potential for full automation. However, these assays have a limited analytical measurement range (AMR) above which antigen excess leads to falsely low estimates of the analyte concentration (i.e., the hook effect). Traditional methods for correction of antigen excess require sample dilution, compromising time and cost-efficiency. Therefore, novel methods that extend the AMR are needed. METHODS: A kinetic model of a generic homogeneous turbidimetric immunoassay was built and then parameterized using a genetic algorithm. Kinetic features that could be used to extend the AMR were identified and subsequently validated with clinical data from consecutive measurements of 2 homogeneous turbidimetric immunoassays: κ serum free light chain and rheumatoid factor. RESULTS: A novel kinetic parameter, the area under the curvature (AUCU), was derived that increases in proportion to the analyte concentration in a range beyond the AMR of conventional end point methods. When applied to clinical data, the AUCU method provided a log-linear calibration curve in the zone of antigen excess extending the AMR by >10-fold for 2 different immunoassays. CONCLUSIONS: The AUCU method detects and corrects antigen excess, extending the AMR in homogeneous turbidimetric immunoassays. The advantage of this method over conventional methods would be a reduction in the number of repeated samples, resulting in significant time and cost savings.

Assessment of Hereditary Thrombophilia: Performance of Protein S (PS) Testing.

Protein S (PS) is a Vitamin K-dependent protein that functions as a cofactor for the regulation of the coagulation system. PS works in conjunction with Activated Protein C to inactivate factors V and VIII. PS circulates in plasma either complexed to the complement protein, C4b Binding Protein or unbound. The unbound (or free) component is the functional form for the regulation of the coagulation system. PS can be measured in plasma by functional activity, the free (or unbound form) or both free and bound fractions (Total PS). The test most widely used for clinical evaluations is the Free PS Antigen assay (which is the surrogate of PS anticoagulant activity) and represents the protocol described in this chapter. The Free PS Antigen assay is an immunologic assay which specifically measures the unbound fraction of PS in test plasma. Other methods for assessing PS are also available, including PS activity and total PS Antigen assays, but protocols for these assays are not provided.

Performance characteristics of an automated latex immunoturbidimetric assay [HemosIL® HIT-Ab(PF4-H)] for the diagnosis of immune heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-PF4/heparin antibodies. Given time-sensitive treatment considerations, a rapid and accurate laboratory test for HIT antibodies is needed. AIMS: To determine operating characteristics for the HemosIL® HIT-Ab(PF4/H), a rapid, on-demand, fully-automated, latex immunoturbidimetric assay (LIA), for diagnosis of HIT. METHODS: We evaluated LIA sensitivity, specificity, negative (NPV) and positive predictive value (PPV), negative (LR-) and positive likelihood ratio (LR+), using citrated-plasma from 429 patients (prospective cohort study of 4Ts scoring; HIT, n=31), and from consecutive HIT patients (n=125), using reference standard serotonin-release assay (SRA). Comparators included two PF4-dependent enzyme-immunoassays (EIAs). We used stratum-specific likelihood ratios (SSLRs) to determine how differing magnitudes of LIA-positivity influenced post-test probability of HIT. RESULTS: LIA operating characteristics were: sensitivity=97.4% (152/156); specificity=94.0% (374/398); PPV=55.6% (30/54); and NPV=99.7% (374/375). At manufacturers' cutoffs, LIA specificity and PPV were superior to the EIAs. Although a negative LIA pointed strongly against HIT (LR-, 0.034), the post-test probability was ~2% with high 4Ts score. The LIA's LR+ was high (16.0), with SSLRs rising substantially with greater LIA-positivity: 5.7 (1.0-4.9U/mL), 31 (5.0-15.9U/mL), and 128 (≥16U/mL). A LIA-positive result (at 1.0 cutoff) indicated at least 24% HIT probability (low 4Ts score), rising to 90% with high 4Ts score. CONCLUSIONS: Although approximately 1 in 40 SRA-positive patients tested LIA-negative, the LIA's high NPV and PPV indicate that this rapid assay is useful for the diagnostic evaluation of HIT, including in low pre-test situations.