Résumé
Objective To improve the reliability and accuracy of WBC counting in cerebrospinal fluid (CSF) ,this article is stud-ying the improved method of WBC counting in CSF by finding out the optimum percentage of CSF specimen with the most suitable concentration of acetic acid .Methods CSF specimen was mixed with different acetic acid at different ratio respectively .WBC counts were performed in 5 minutes on diluted samples of various concentrations .A series of 20 CSF specimens were analyzed via the proposed assay and conventional method .The average value and coefficient of variation (CV) of WBC count of each sample were c compared and analyzed .Results The optimum percentage of CSF sample was obtained at 60∶40 ratio .In this percentage , the maximal WBC count (189/μL) was obtained compared that of conventional method (161/μL) .Moreover ,the CV of the WBC counts in this percentage (7% ) was also lower than that of the conventional method (18% ) .Conclusion The reliability and accur-ancy of WBC counting in CSF was the optimum percentage of CSF specimen and 5% acetic acid was 60 :40 .It may lead to a more reliable ,accurate and standard way of WBC counts in CSF .
Résumé
One highly pathogenic strain of avian influenza virus (AIV) was isolated from goose in China recently, designated as F-3. In order to study the viral entry mechanisms, the hemagglutinin (HA) gene of H5N1 subtype AIV isolate was amplified by RT-PCR, and then cloned into pGEM-T vector and sequenced. The sequencing result has logging in GenBank, the accession number was AY639405. The HA gene of F-3 had a complete open reading frame (ORE) and composed of 1707 nucleotides, coding for 568 amino acids. The deduced amino acid sequence at the cleavage site of the HA protein was RKKR GLF, matched to the characteristic of virulent avian influenza strain. The HA gene were subcloned into pcDNA3, so the plasmid pcDNA-HA can express the HA glycoprotein. Co-transfected pcDNA-HA, pHIT60 (include Murine Leukemia Virus structural genes, namely gag and pol) and pHIT111 (retroviral vector genome,containing LacZ as a reporter) into 293T cells. The retroviral supernatant were harvested 48 hours post-transfection, filtered through 0.45 micromol/L filter. The supernatant were used to analysis the characteristic of the pseudotyping virions by Western blotting and infection test. Western blotting revealed the HA glycoproteins can be expressed on the virions, indicated the glycoproteins were incorporated onto the retroviral virions. Infection test were performed on 293T, NIH3T3 and COS-7, all the three kinds of cells infected were lacZ positive, indicating viral entry, and revealed the pseudotype virions of MuLV-HA were infectious. So the pseudotype system of MuLV particles with AIV Hemagglutinin proteins were setted up and it can be used to study the entry of avian influenza virus isolated from goose in China.