Matrix-normalised quantification of species by threshold-calibrated competitive real-time PCR: allergenic peanut in food as one example.

A novel method to quantify species or DNA on the basis of a competitive quantitative real-time polymerase chain reaction (cqPCR) was developed. Potentially allergenic peanut in food served as one example. Based on an internal competitive DNA sequence for normalisation of DNA extraction and amplification, the cqPCR was threshold-calibrated against 100mg/kg incurred peanut in milk chocolate. No external standards were necessary. The competitive molecule successfully served as calibrator for quantification, matrix normalisation, and inhibition control. Although designed for verification of a virtual threshold of 100mg/kg, the method allowed quantification of 10-1,000 mg/kg peanut incurred in various food matrices and without further matrix adaption: On the basis of four PCR replicates per sample, mean recovery of 10-1,000 mg/kg peanut in chocolate, vanilla ice cream, cookie dough, cookie, and muesli was 87% (range: 39-147%) in comparison to 199% (range: 114-237%) by three commercial ELISA kits.

Detectability of lupine seeds by ELISA and PCR may be strongly influenced by potential differences between cultivars.

Accurate methods for allergen detection are needed for the verification of allergen labeling and the avoidance of hidden allergens. But systematic data on the influence of different cultivars of allergenic crop species on their detectability in enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are lacking. As one example, seeds of 14 different cultivars of lupine (Lupinus albus, Lupinus angustifolius, Lupinus luteus) were investigated for total protein according to a Kjeldahl method, and for their relative quantitative detectability in three commercial lupine-specific ELISA tests and four lupine-specific PCR methods. Total Kjeldahl nitrogen allowed an accurate quantification of total protein. Relative differences in quantitative response between cultivars of 390-5050% and 480-13,600% were observed between ELISA kits and PCR methods, respectively. Hence, quantitative results of selected ELISA and PCR methods may be strongly influenced by the examined lupine cultivar.

Evaluation of Quality Parameters for Cord Blood Donations.

SUMMARY: BACKGROUND: Umbilical cord blood (CB) is widely used for hematopoietic stem cell transplantation and holds promise for the development of innovative medicinal products. In order to find out whether the conditions for collection and storage before processing might have an impact on the quality of CB preparations, viability and the clonogenic potential were assessed. METHODS: CB was collected under field conditions. Flow cytometry was used to determine leukocytes, CD34/CD45+ cells, viability, and nucleated red blood cells (NRBC). Clonogenic activity was determined using isolated mononuclear cells (MNC). RESULTS: Neither plasma citrate concentrations nor storage temperature (within 24 h) affected cell viability or colony formation. After storage for 49-80 h, leukocyte viability declined by about 16% compared to CB stored up to 24 h. In contrast, the clonogenic activity and CD34/CD45+ cell content were not affected. A higher gestational age was associated with a lower yield of clonogenic activity compared to midterm deliveries. NRBC varied widely (median 7.3%; range 0.63-17.3%) without relation to gestational age or colony formation. There was a close correlation between the percentage of viable CD34/CD45+ cells and colony formation (r = 0.77 for CFU-GM; r = 0.75 for CFU-C). CONCLUSIONS: The content of viable CD34/CD45+ cells represents the clonogenic activity of CB preparations. Therefore, determination of viable CD34/CD45+ cells should be generally performed as a routine quality control assay.