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Cytomegalovirus (CMV) infection is one of the most important infectious complications of transplantation. Monitoring CMVspecific CD8 T cell immunity is useful for predicting activeCMVinfection and for directing targeted antiviral therapy. In this study, we examined four basic parameters for validation of CMV-specific tetramer staining and peptide stimulation assays that cover five most frequent HLA class I alleles. We also examined the potential use of CMV-specific CD8+ T cell numbers and functional and cytolytic responses in two autologous HSCT recipients treated for multiple myeloma. The coefficient of variation (CV %) of the precision within assays was 3.1-24% for HLA-tetramer staining, 2.5-47% for IFN-γ, and 3.4-59.7% for CD107a/b production upon peptide stimulation. The precision between assays was 5-26% for tetramer staining, 4-24% for IFN-γ, and 5-48% for CD107a/b. The limit of detection was 0.1-0.23 cells/μL of blood for tetramer staining, 0-0.23 cell/μL for IFN-γ, and 0.11-0.98 cells/μL for CD107a/b. The assays were linear and specific. The reference interval with 95% confidence level was 0-18 cells/μL for tetramer staining, 0-2 cells/μL for IFN-γ, and 0-3 cells/μL for CD107a/b. Our results provide acceptable measures of test performance for CMV immune competence assays for the characterization of CD8+ T cell responses posttransplant measured in the absolute cell count per μL of blood [ABSTRACT FROM AUTHOR]

Background: 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., Methods: We ran 109 healthy donor EDTA plasmas and 80 sera samples with both factor B immunodiffusion (The Binding Site) and Quidel Bb EIA assays., Results: 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., Conclusions: 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.

Background: 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., Methods: 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., Results: 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., Conclusions: For TGF-beta1 assay, urine sample preparation should not include a centrifugation step; repeating freeze-thawing should be avoided.