Igor Y. Pavlov

During the alternative complement pathway activation, factor B is cleaved in two fragments, Ba and Bb. Concentration of those fragments is about 2 logs lower than of factor B present in the blood, which makes fragment detection challenging because of potential cross-reactivity. Lack of information on Bb assay cross-reactivity stimulated the authors to investigate this issue. We ran 109 healthy donor EDTA plasmas and 80 sera samples with both factor B immunodiffusion (The Binding Site) and Quidel Bb EIA assays. During the study it was shown that physiological concentrations of gently purified factor B demonstrated approximately 0.15% cross-reactivity in the Quidel Bb EIA assay. We also observed that Bb concentration in serum is higher than in plasma due to complement activation during clot formation which let us use sera as samples representing complement activated state. Our study demonstrated that despite the potential 0.15% cross-reactivity between endogenous factor B and cleaved Bb molecule, measuring plasma concentrations of factor Bb is adequate to evaluate the activation of the alternative complement pathway.

TGF-beta1 is a mediator of immune and anti-inflammatory responses. The present research was dedicated to the validation of the urine Quantikine Human TGF-beta1 ELISA assay. A reference interval was developed utilizing 100 urine samples from healthy donors. Samples containing precipitate were centrifuged and supernatants or un-spun specimens were run with TGF-beta1 assay. Supernatant TGF-beta1 concentrations are only a fraction of the un-spun sample values (77% to 29%, even less for the low concentration samples). Validation involving un-centrifuged urine samples demonstrated the following TGF-beta1 assay characteristics: linearity from 23 to 1286 pg/mL, recovery from 95 to 116%; precision: < 17%; analytical specificity 3 pg/mL; reference interval from 0 to 39 pg/mg creatinine; freeze-thaw stability: after first thawing, urine samples could loose from 5 to 40% of TGF-beta1 activity on each following freeze-thaw circle. For TGF-beta1 assay, urine sample preparation should not include a centrifugation step; repeating freeze-thawing should be avoided.

Reference intervals (RI) play a key role in clinical interpretation of laboratory test results. Numerous articles are devoted to analyzing and discussing various methods of RI determination. The two most widely used approaches are the parametric method, which assumes data normality, and a nonparametric, rank-based procedure. The decision about which method to use is usually made arbitrarily. The goal of this study was to demonstrate that using a resampling approach for the comparison of RI determination techniques could help researchers select the right procedure. Three methods of RI calculation—parametric, transformed parametric, and quantile-based bootstrapping—were applied to multiple random samples drawn from 81 values of complement factor B observations and from a computer-simulated normally distributed population. It was shown that differences in RI between legitimate methods could be up to 20% and even more. The transformed parametric method was found to be the best method for the calculation of RI of non-normally distributed factor B estimations, producing an unbiased RI and the lowest confidence limits and interquartile ranges. For a simulated Gaussian population, parametric calculations, as expected, were the best; quantile-based bootstrapping produced biased results at low sample sizes, and the transformed parametric method generated heavily biased RI. The resampling approach could help compare different RI calculation methods. An algorithm showing a resampling procedure for choosing the appropriate method for RI calculations is included.

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G againstLeishmaniaspecies. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported.