Measurement of CD83 mRNA by real-time polymerase chain reaction to determine removal of dendritic cells by leucoreduction of whole blood.
Information is lacking regarding efficiency of removal of circulating dendritic cells (DCs) by leucoreduction (LR) of blood. This is important since DCs may play a role in transporting abnormal prion, the likely infectious agent of variant Creutzfeldt-Jakob disease. In this study, we report development of a real-time polymerase chain reaction (RT-PCR) assay to quantify residual DCs in LR whole blood via measurement of selected messenger RNA (mRNA) markers. Taqman-based RT-PCR assays were set up for CD83 as a marker of mature DCs, and CD1c, CD11c, CD303 and CD304 as markers for plasmacytoid and myeloid DCs along with the pan-leucocyte marker CD45. We then assayed 46 paired pre-/post-LR whole blood samples and determined the log(10) reduction of their CD83 and CD45 mRNA. Our data indicate that RT-PCR can be used to detect suitably low CD83 mRNA levels. We measured a median log(10) reduction for CD83 mRNA of 4.5 [standard error of the mean (SEM) 0.07] and 4.1 (SEM 0.10) for CD45 mRNA. These reductions are comparable to cell removal, where flow cytometry indicated a reduction in total white cell counts of 4.3 log(10) (SEM 0.09). Our other group of markers were reduced to their detection limits, CD1c (3.9 log(10), SEM 0.3), CD11c (5.0 log(10), SEM 0), CD303 (3 log(10), SEM 0.1), CD304 (4.0 log(10), SEM 0) which are all higher than the minimum specifications for LR products. In conclusion, we successfully developed an RT-PCR assay to quantify suitably low numbers of DC cells. We show for the first time that DCs are effectively removed using a standard whole blood filter.
Morrison
Alex
A
Products & Components R & D Group, National Science Laboratory, Scottish National Blood Transfusion Service, Edinburgh, UK. alex.morrison@snbts.csa.scot.nhs.uk
Macgregor
Ian
I
D015703:Antigens, CD / Q000235:genetics*; D002469:Cell Separation / Q000379:methods; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D017493:Leukocyte Common Antigens / Q000235:genetics; D007962:Leukocytes / Q000166:cytology*; D008562:Membrane Glycoproteins / Q000235:genetics*; D012313:RNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D000097802:CD83 Antigen
D015703:Antigens, CD; D007136:Immunoglobulins; D017493:Leukocyte Common Antigens; D008562:Membrane Glycoproteins; D012313:RNA; D012333:RNA, Messenger; D000097802:CD83 Antigen