Su.94. Phenotypic Differences Between Cord Bloodand Adult Peripheral BloodMaria Lopez, David Lawrence. Wadsworth Center, Albany,NY

It is well known that neonates are more susceptible toinfections than adults, and it is generally assumed thatneonates have a more immature immune system. Theobjective of this study was to compare cord blood (CB) andadult peripheral blood (APB) to establish whether or not thereare immunophenotypical differences. CB and APB werecollected into CPD anticoagulant. Whole blood was stainedwith fluorochrome conjugated antibodies, cells were fixed,red cells were lysed and samples were run in a FACSCanto flowcytometer using FACSDiva software. When data were ana-lyzed, plots of SSC vs. CD45 showed two lymphocyte-likepopulations in CB with the same low SSC characteristics, whilethere was only one low SSC lymphoid population in APB. TheCD45dim population included the majority of CD34+ cells,but it also included T, B, NKT and NK cells. The percentagesof CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD3+CD56+ and CD3-CD56+ in the CD45high gate of CB were similar to thepercentages obtained for APB. Meanwhile, in the CD45dimgate, the percentages were either lower (CD3+, CD3+CD4+,CD3+CD8+) or higher (CD19+, CD3+CD56+, CD3-CD56+) than inthe CD45high gate. Therefore, this study indicates that CBpresents two lymphoid populations defined by CD45 expres-sion; the CD45dim subsets likely represent cells that haveelevated proliferative activity due to low phosphataseexpression, but that are still not yet immunologically maturewith regard to immunophenotype and function.

doi:10.1016/j.clim.2008.03.445

Su.95. HLA Class I and Class II Allele and HaplotypeDistribution in the Venezuelan PopulationMaría del Pilar Fortes, María Paredes, Gisselle Gil, MarinaPalacios, Blanca Isaac, Paolo Tassinari. Institute ofImmunology (FOCIS Center of Excellence), UniversidadCentral de Venezuela, Caracas, Venezuela

Population studies represent an integral part and a necessarylink in a complex chain of host-pathogen interactions, diseasepathogenesis, and major histocompatibility complex poly-morphism. Genes of Mongoloid, Negroid, and caucasoid originhave created a distinctive human leukocyte antigen (HLA)genetic profile in the Venezuelan population that will influenceHLA and disease association studies. Our objective was todeterminate the predominant alleles and haplotype frequenciesin this hybrid mestizo population. The population consisted of486 unrelated healthy Venezuelan mestizos individuals .Weexamined the frequency of HLA A-B-C, HLA-DQ and HLA-DRgenes by polymerase chain reaction amplification and sub-sequent hybridization with sequence-specific oligonucletideprobes. Phenotypic and allelic frequencies were calculated bydirect counting. Haplotype frequencies were calculated bymeans of maximum likehood estimates of gene counting. Indecrescent order, the most frequent alleles of HLA class I wereA⁎02, A⁎24, A⁎68, B⁎35, B⁎44, B⁎51, B⁎07, B⁎15 and Cw⁎07. ForHLA class II, the most frequent alleles were DQB1⁎03 and

DRB1⁎04, DRB1⁎15, DRB1⁎13, DRB1⁎07. The predominanthaplotype was HLA- A⁎02 B⁎35 DQB1⁎03 DRB1⁎04. Some ofthese alleles and haplotypes frequencies are predominantlypresent among amerindians living in Venezuela (A⁎02, A⁎24,B⁎35, Cw⁎07, DRB1⁎04, A⁎24 B⁎35), and has been reported witha high incidence in several European populations (A⁎02, B⁎44,B⁎51, DRB1⁎15, DRB1⁎13, DRB1⁎07) and African Americans(A⁎68, B⁎07, B⁎15), reflecting the native population's admix-ture. A detailed analysis of HLA class I and II allelepolymorphism in the Venezuelan population confirms thehistorically known influence of genes of Mongoloid, Negroidand Caucasoid origin.

doi:10.1016/j.clim.2008.03.446

Su.96. High Throughput PBMC (Peripheral BloodMononuclear Cells) Isolation Steps Using the BiomekSeries of Automated Liquid HandlersCarlos Aparicio, Edward Jachimowicz, Mahsa Aspsater,Ileana Munoz-Antoni, Enrique Rabellino. Beckman Coulter,Miami, FL

Purification of PBMC is a laborious and variable manualprocess and, thus, one of the limiting factors forprocessing patient specimens in large clinical trials usingdifferent test sites across the world. Our study focuses onan automated strategy using the Ficoll-Paque™ densitygradient as the central technology to isolate PBMC fromwhole blood samples. The process utilizes the Biomek FXliquid handler to minimize the number of steps andoperator interventions in current laboratory procedures.The automated strategy relies on under-laying the densitygradient in diluted whole blood, centrifugation, andharvesting the cells from a pre-determined height in thecentrifugation tubes. Current throughput for Ficoll-Paqueaddition and cell harvesting is less than 20 and 30seconds/tube, respectively, for an overall throughputadaptable to over 50 tubes per hour. PBMC harvested bycurrent manual procedures and the automated processwere compared on the basis of 1) total cell recovery, 2)cell viability, 3) selective cell phenotype recovery, and 4)cell function characteristics pre- and post-freeze/thawstorage. Results showed a) minimum of 88% total cellrecovery compared to the manual method, b) cell viabilitygreater than 93%,and c) phenotype recovery of majorlymphocyte sub-populations were ±5% of values obtainedby manual method. This novel fully automated andstandardized method for preparing PBMC will assist cellbased-assay testing strategies in clinical trials by improv-ing overall quality and efficiency of the process as well asreducing labor and cost.

doi:10.1016/j.clim.2008.03.447

Su.97. Inhibitory Regulation of CEACAM1 on T CellReceptor and NKG2D Mediated SignalingZhangguo Chen, Lanfen Chen, Shuo-Wang Qiao, TakashiNagaishi, Richard Blumberg. Brigham and Women's Hospital,Harvard Medical School, Boston, MA

S155Abstracts