VACUTAINER CPT and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples.
BACKGROUND
For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNgamma expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery.


RESULTS
The results indicate that cryopreserved PBMC samples tested for CMV- and HIV-specific interferon-gamma (IFNgamma) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNgamma response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT.


CONCLUSIONS
These data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV+ PBMC, as well as a viable alternative to Ficoll gradient separation.
Ruitenberg
Joyce J
JJ
BD Biosciences, San Jose, CA, USA. joyce_ruitenberg@bd.com
Mulder
Candice B
CB
Maino
Vernon C
VC
Landay
Alan L
AL
Ghanekar
Smita A
SA
D001793:Blood Preservation; D001800:Blood Specimen Collection / Q000295:instrumentation*; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D002986:Clinical Trials as Topic / Q000379:methods; D015925:Cryopreservation; D003587:Cytomegalovirus / Q000276:immunology; D005362:Ficoll; D005434:Flow Cytometry / Q000379:methods; D015683:Gene Products, gag / Q000276:immunology; D006679:HIV Seropositivity / Q000097:blood*; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000097:blood; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008213:Lymphocyte Activation; D010750:Phosphoproteins / Q000276:immunology; D011498:Protein Precursors / Q000276:immunology; D014763:Viral Matrix Proteins / Q000276:immunology
D015683:Gene Products, gag; D010750:Phosphoproteins; D011498:Protein Precursors; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D005362:Ficoll; D007371:Interferon-gamma