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A double-antigen sandwich ELISA was developed for the detection of antibodies to influenza A viruses. A recombinant nucleoprotein (rNP) of influenza A virus was used as a capture antigen and an HRP-conjugate for detecting the antibodies. A total of 125 serum samples from birds of different species including chickens, geese, open-billed storks, Khaki Campbell ducks, lesser whistling ducks, and pigeons with known antibodies were tested by ELISA. The sensitivity and the specificity of ELISA were found to be 98% and 97.3%, respectively. The assay was able to detect the presence of influenza A antibodies as early as the fourth day post-inoculation in ducks infected experimentally with influenza A (H5N1) virus. Excellent agreement (97.6%) was obtained between this sandwich ELISA and the hemagglutination inhibition (HI) tests (kappa=0.95). The double-antigen sandwich ELISA correlated well with a commercial avian influenza (AI) multispecies ELISA and was slightly more sensitive than the AI multispecies ELISA. These findings indicate that the double-antigen sandwich ELISA based on rNP may offer an effective screening method for serodiagnosis of influenza A virus. The double-antigen sandwich ELISA also enables the detection of antibodies to influenza A viruses in different species without the need for species-specific secondary antibodies., (2009 Elsevier B.V. All rights reserved.)

Umbilical cord blood (UCB) is being increasingly used as an alternative source of hematopoietic stem cells for allogeneic bone marrow transplantation. UCB transplantation has been successfully used to treat a variety of genetic, hematological, and oncological disorders in children and adults. The objectives of this study was to establish a closed-system technique for UCB collection and buffy coat separation by Optipress I device. Thirty-four UCB were collected by triple-bag system from pregnant mothers whose fetuses were not affected by thalassemic diseases after prenatal diagnosis. The mean volumn of UCB collection were 120 +/- 5 ml (range 65-180 ml). Total WBC, CD34+ cells, the progenitor cell erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) in the UCB units were (9.36 +/- 0.84) x 10(8), (3.61 +/- 0.52) x 10(6), (9.12 +/- 1.60) x 10(5), and (5.32 +/- 1.23) x 10(5), respectively. Good correlation between the nucleated cell and net cord blood volume could be demonstrated (p < 0.0001). The correlation between CD34+ cells and the following parameters: nucleated cell, BFU-E or CFU-GM were also demonstrated (p = 0.001, 0.0105 or 0.0001, respectively). Buffy coat was subsequently separated from 18 UCB units by Optipress I device. 70 +/- 3 ml of buffy coat were collected and cryoprocessing was done by automatic controlled-rate freezer. Good recovery of total WBC, CD34+ cells, progenitor cells BFU-E and CFU-GM after buffy coat separation were observed 89 per cent, 95 per cent, 109 per cent, and 102 per cent respectively. There was no aerobic bacterial or fungal contamination in the separated blood products. By using this technique, the UCB units were easily collected, rapidly separated within one hour, and high recovery of the hematopoietic progenitor cells could be obtained.

Very limited data exists in Thailand regarding the frequency of BCR-ABL leukemic gene and its prognostic implication in Thai CML patients. The objective of this study was to develop a rapid molecular assay for the detection of the two most commonly reported variants of BCR-ABL fusion gene, B2A2 and B3A2 in CML patients. Bone marrow or peripheral blood were used for RNA extraction and reverse-transcribed to cDNA for PCR amplification. 92 per cent of CML patients (91/99) were positive for BCR-ABL gene (61% B3A2 and 31% B2A2). 8/99 CML patients were BCR-ABL-negative. B3A2 and B2A2-positive patients did not have any different clinical and hematological features at presentation although B3A2 patients tended to be slightly older and had higher platelet counts. 71/71 non-CML including other MPD and leukemia cases were all negative for BCR-ABL gene. In conclusion, a rapid RT-PCR assay has now been developed for the detection of this hallmark gene in CML patients. It should be of great value in the differential diagnosis of CML from other diseases. Long-term follow-ups of CML patients with different variants are needed to determine the prognostic importance of each gene variant.