Provision of Epstein‐Barr virus‐transformed B‐cell lines in a routine tissue typing laboratory: practicalities and applications

Abstract

A bank of Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BCLs) has been established in this laboratory over the last 7 years. Novel, rare and unusual HLA phenotypes were highlighted during routine clinical testing and during typing of volunteer haematopoietic stem cell donors for the Welsh Bone Marrow Donor Registry. The BCLs are routinely used as laboratory reagents, as a source of DNA for reference purposes and for research work. Consenting donors are tested for hepatitis B surface antigens (HBsAg), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) before their B-lymphocytes are transformed. Strict bio-identity checks are performed before and after transformation and after each BCL expansion. Each BCL is tested regularly for mycoplasma contamination. A total of 230 blood samples were transformed. One hundred and fifty-nine sterile samples produced 157 BCLs (98.7% success), while 71 non-sterile samples produced 50 BCLs (70.4% success), giving an overall success rate of 90.0%. Fifteen of the transformation failures have since been repeated successfully. Factors contributing to the high success rate and reasons for the 23 failed transformations are discussed. The successful development and use of pools of BCLs for HLA antibody screening by flow cytometry are described. Whilst certain training and health and safety issues require close attention, it is clear that EBV transformation of B lymphocytes and/or the use of BCLs is feasible in a routine tissue typing laboratory and that BCLs are a valuable resource. Careful adherence to the methods and procedures detailed here should virtually guarantee successful transformation (98.7%) from good quality, sterile whole blood samples.

A “silent” nucleotide substitution in exon 4 is responsible for the “alternative expression” of HLA-A*01:01:38L through aberrant splicing

“…PBLs were obtained by standard density gradient centrifugation methods and BCLs were prepared using our previously published methods [20]. All control cells were HLA-A, -B, and -C typed by PCR-SSP [17] and standard serology [21].…”

Human Lymphoblastoid Cell Lines in Pharmacogenomics

“…Factors contributing to the high success rate of EBV transformation include: processing in a sterile environment against microbial contamination; protecting cross-contamination of cell lines; using high-potency viral supernatant of sufficient titer; using good-quality blood samples (e.g., sufficient number of lymphocytes more than 10 6 , not contaminated with red blood cells) [20]. Factors contributing to the high success rate of EBV transformation include: processing in a sterile environment against microbial contamination; protecting cross-contamination of cell lines; using high-potency viral supernatant of sufficient titer; using good-quality blood samples (e.g., sufficient number of lymphocytes more than 10 6 , not contaminated with red blood cells) [20].…”

HLA‐B*15:33, a rare allele whose product reacts as an HLA‐B62 and ‐Cw5/Cw8 specificity

“…An Epstein–Barr virus transformed B‐cell line (Bass et al. , 2004) is available from the B*15:33 donor.…”