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Although the benefits of quantifying serum squamous cell carcinoma antigen (SCCa) have been reported, SCCa reagents were no longer available in the US by the late 1990s. Because SCCa quantification still has demonstrated clinical utility, we developed and validated a microtiter plate–based ELISA for measuring SCCa in serum.

We coated microtiter strips overnight with capture anti-SCCa monoclonal antibody, washed the wells, added blocking buffer, and lyophilized the strips. For detection, we used a biotinylated anti-SCCa detection antibody, streptavidin/horseradish peroxidase conjugate, and tetramethylbenzidine/H₂O₂ substrate. A novel blocking reagent against human antimouse antibodies (HAMA) was evaluated. A reference interval was established with sera from healthy individuals and was confirmed in smokers.

The assay was linear to 40 µg/L SCCa (slope, 1.00; y intercept, 0.695; R², 0.996) with a detection limit of 0.3 µg/L. The intraassay imprecision results [mean (CV)] were 2.5 µg/L (3.4%), 18.0 µg/L (3.0%), and 30.7 µg/L (2.4%); interassay imprecision results were 2.0 µg/L (9.9%), 20.0 µg/L (7.6%), and 36.3 µg/L (3.5%). A correlation analysis against an established automated assay generated a slope of 0.976 and a y intercept of –0.193 µg/L (r² = 0.916). An upper reference limit of 2.1 µg/L SCCa was established at 95% confidence level, with no difference observed in smokers. No correlation between SCCa concentration and age was observed (r² = 0.0003). At a blocking reagent concentration of 5 mg/L, HAMA interference was eliminated in 3 samples known to produce falsely increased SCCa results.

This SCCa ELISA demonstrates acceptable performance characteristics for quantifying serum SCCa and is effective in eliminating HAMA interference.

Coding mutations in the AR (androgen receptor) gene have been identified in tissue samples from patients with advanced prostate cancer and represent a possible mechanism underlying the development of castration-resistant prostate cancer (CRPC). There is a paucity of tumor-derived tissue available for molecular studies of CRPC patients. Circulating tumor cells (CTCs) in the blood of CRPC patients represent a possible avenue for interrogating the disease of such patients.

Circulating tumor cells were captured with the CellSearch® Circulating Tumor Cell (CTC) Kit and with the CellSearch Profile Kit plus Qiagen's AllPrep DNA/RNA Micro Kit for the measurement of the CTC count per 7.5 mL of blood and for the isolation of nucleic acids, respectively. The AR gene was amplified by the PCR, and mutation status and relative abundance were analyzed by applying Transgenomic's WAVE® denaturing HPLC technology followed by direct sequencing.

AR mutations were detected in 20 of 35 CRPC patients; 19 missense mutations, 2 silent mutations, 5 deletions, and 1 insertion were observed. The relative abundance of the mutants in the amplified products ranged from 5% to 50%. Many of the AR mutations were identified in surgical biopsies or at autopsy and were associated with resistance to androgen-directed therapies.

AR mutations can be identified in CTC-enriched peripheral blood samples from CRPC patients. This approach has the potential to open new perspectives in understanding CTCs and the mechanisms for tumor progression and metastasis in CRPC.

Biomarker assays are often conducted on whole blood samples in the course of drug development studies. Because bacterial lipopolysaccharide (LPS) (endotoxin) contamination is known to cause spontaneous cytokine production by monocytes, contamination of blood collection tubes may interfere with biomarker assay results.

Whole blood from healthy donors was collected into plastic or glass sodium (Na⁺)-heparin Vacutainer^TM blood collection tubes and heparinized syringes. Samples were analyzed for phosphoprotein response, cytokine production, and RNA expression. Tubes were tested for endotoxin contamination by use of the limulus amoebocyte lysate assay.

Results of phospho-flow cytometry, branched DNA (bDNA), and ELISA assays indicated that a specific lot (#5339582) of plastic Na⁺-heparin Vacutainer tubes was highly contaminated with an endotoxinlike substance, and contamination was confirmed by the limulus amoebocyte lysate assay. Analysis of multiple-analyte panels revealed that analytes whose changed expression was predictive of LPS stimulation were increased when whole blood was incubated in contaminated tubes for 6 or 18 h. Two additional lots of plastic tubes tested had detectable amounts of endotoxin sufficient to strongly alter phospho-flow cytometry analyses, as determined by the fold change in phosphorylation of p38 mitogen-activated protein kinase in response to tumor necrosis factor and LPS. In contrast, 3 lots of glass tubes had substantially lower levels of spontaneous blood activation.

Endotoxin contamination associated with tubes from 3 lots of a particular type of plastic Na⁺-heparin Vacutainer tube dramatically affected biomarker assay measurements. Prescreening these tubes is suggested before their use in clinical sample analysis.

Standardization of clinical measurements of C-reactive protein (CRP) is based on the availability of reference materials. The serum protein reference material ERM-DA470 was used as a calibrant for various commercially available immunoassays but has now been exhausted. The recently released ERM-DA470k/IFCC was intended to fully replace ERM-DA470. However, the new material was not suited for the certification of CRP because of a bias introduced by the lyophilization process that caused loss of about 20% of CRP measurable by routine immunoassays, compared with the nonlyophilized material that was stored in a liquid frozen state.

We investigated the physicochemical state of CRP in a set of 4 lyophilized and 2 nonlyophilized serum-based CRP-containing materials by semi-native gel electrophoresis, Western blotting, and gel filtration.

We detected a monomeric form of CRP (mCRP) in lyophilized materials at a concentration significantly higher than seen in the materials not subjected to lyophilization. Different reconstitution protocols led to variations of the monomeric CRP fraction found in reconstituted, previously lyophilized material.

Most of the 20% loss in measured CRP after lyophilization of the material can be accounted for by the dissociation of natively pentameric CRP into subunits. The observed dissociation results from lyophilization and subsequent reconstitution of the material at insufficient concentration levels of calcium ions. In the presence of various protein forms, differences in antibody specificity and reactivity between immunoassays and alterations of stoichiometry of antigen–antibody interactions can contribute to the divergence of the measured values.

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell–receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample.

We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded.

The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR.

Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Severe combined immunodeficiency (SCID) fulfills many of the requirements for addition to a newborn screening panel. Two newborn screening SCID pilot studies are now underway using the T-cell receptor excision circle (TREC) assay, a molecular technique. Here we describe an immunoassay with CD3 as a marker for T cells and CD45 as a marker for total leukocytes that can be used with the Guthrie specimen.

The multiplexing capabilities of the Luminex platform were used. Antibody pairs were used to capture and detect CD3 and CD45 from a single 3-mm punch of the Guthrie specimen. The assay for each biomarker was developed separately in identical buffers and then combined to create a multiplex assay.

Using calibrators made from known amounts of leukocytes, a detection limit of 0.25 x 10⁶ cells/mL for CD3 and 0.125 x 10⁶ cells/mL for CD45 was obtained. Affinity tests showed no cross-reactivity between the antibodies to CD3 and CD45. The multiplex assay was validated against 8 coded specimens of known clinical status and linked to results from the TREC assay that had identified them. All were correctly identified by the CD345 assay.

The performance parameters of the CD345 assay met the performance characteristics generally accepted for immunoassays. Our assay classifications of positive specimens concur with previous TREC results. This CD345 assay warrants evaluation as a viable alternative or complement to the TREC assay as a primary screening tool for detecting T-cell immunodeficiencies, including SCID, in Guthrie specimens.