[Residual leukocyte counting in plasma by a real-time genomic amplification assay].
BACKGROUND
Systematic plasma leukoreduction, which was introduced in France in April 1st 2001, has given rise to more sensitive methods for residual leukocytes counting. The technologies in application at this moment (Nageotte hemocytometers and flow cytometry methods) have been modified by a thirty fold sample concentration prior to analysis, inducing frequent downgrading of the plasma unit. So, in order to improve the detection threshold, we developed a more sensitive assay using "Real-Time Polymerase Chain Reaction" technology.


METHODS
Real-time polymerase chain reaction was performed on a highly conserved HLADQalpha1 gene sequence. In order to determine the analytical performances of the method (accuracy, sensitivity, linearity and specificity) serial dilutions series ranging from 10(4) to 1 cells/ml were performed. A total of 18 series were prepared from three leukocyte stock solutions, by 1 in 10 serial dilutions in three plasmas completely devoided of white cells (called negative plasmas). To examine the specificity of the assay two negative controls were analyzed in each run.


RESULTS
a sensitivity of 10 cells/ml (10(4) leukocytes/l) was achieved and the assay was linear between 10 and 10(4) cells/ml. The slope (-3.72) of the average standard curve calculated from all series, showed an amplification yield of 92.85%.


CONCLUSIONS
we developed a quantitative assay for residual leukocytes in leukodepleted plasma, that agreed with the quality control requirement specifications.
Segarra
C
C
Laboratoire R&D, EFS Pyrénées-Méditerranée, Montpellier, France. christiane.segarra@efs.sante.fr
Riquet
N
N
Coste
J
J
D052160:Benzothiazoles; D003199:Computer Systems; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D005802:Genes, MHC Class II; D006683:HLA-DQ Antigens / Q000235:genetics; D059848:HLA-DQ alpha-Chains; D006801:Humans; D007958:Leukocyte Count / Q000295:instrumentation / Q000379:methods*; D047589:Leukocyte Reduction Procedures / Q000592:standards; D009930:Organic Chemicals / Q000032:analysis; D010949:Plasma / Q000166:cytology*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011786:Quality Control; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; C069050:HLA-DQA1 antigen; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I