Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique

Intrahepatic lymphocytes (IHL) with their diverse and distinctive subsets emphasise the importance of the liver as a site of immunological activity, but special care is required for their isolation and characterisation. Protocols for IHL isolation, purification and FACS analysis were devised and compared with published extraction protocols. We have reduced the time that IHL are exposed to potentially damaging enzymes during extraction and purified specific subsets using monoclonal antibody (mAb)-coated magnetic microbeads. This has yielded IHL populations with higher viability than previously described protocols (92-95%, compared with 39-86%). Flow cytometric characterisation of IHL subset immunophenotypes was optimised by combining CD45 staining (fluorescence gating) with traditional light scatter properties. Using a panel of mAb and liver biopsies obtained from 23 cadaveric liver transplant donors, we show that the normal liver contains a heterogeneous IHL population with distinctive phenotypes. CD8+ IHL was the predominant population with a mean CD4/CD8 ratio of 1:1.7. Up to 40% of IHL expressed gammadeltaTCR and a third expressed CD56 NK marker; indicating a site of intense immunological activity. The techniques described will allow these cell types to be isolated, fully characterised and their physiological functions to be determined. The histologically normal liver contains heterogeneous and diverse IHL with large numbers of CD8+, NK, NKT and gammadelta+ cells.