Lymphocyte, Granulocyte and Platelet Contamination of Blood Frozen by the Low‐Glycerol Liquid Nitrogen Technique

Summary. The residual leucocyte and platelet content of blood frozen in 20% (v/v) glycerol was assessed by several methods. The final red cell suspension contained 6–12% of the original leucocytes in the form of intact lymphocytes, whilst intact polymorphonuclear cells and platelets could not be detected. The lymphocytes were nonviable as assessed by trypan blue dye exclusion but there was a low response to phytohaemagglutinin stimulation in culture. Using 125I cell‐surface labelling it was found that 39·5 ± 7% (SD) of the labelled lymphocyte surface material, 25·8 ± 6·6% of the granulocyte material and 2·6 ± 0·4% of the platelet surface membrane material remained in the final product after processing. The level of the remaining leucocyte derived material could be lowered by removing the buffy coat after the centrifugal wash steps and by passage through a 40 μan micropore filter; however, this was at the expense of a loss of approximately 25% of red cells. The intact lymphocytes which remained were non‐antigenic in vitro as assessed by the one‐way mixed lymphocyte reaction using the recovered cells as stimulators. However, when the antigen status of the frozen blood as a whole was studied in vivo by injection into laboratory animals, this blood stimulated lymphocytotoxic antibody production, although the levels of antibody were lower and appeared later than those induced by the infusion of a comparable volume of fresh blood. The antibody levels could be diminished further by passing the blood through a 40 μm micropore filter prior to injection.