STANDARDIZATION OF THE HUMAN CYTOMEGALOVIRUS ANTIGENEMIA ASSAY BY MEANS OF IN VITRO-GENERATED PP65-POSITIVE PERIPHERAL-BLOOD POLYMORPHONUCLEAR LEUKOCYTES

We generated in vitro human cytomegalovirus (HCMV) pp65-positive polym orphonuclear leukocytes (PMN) resembling those detected in vivo, follo wing cocultivation of PR IN from healthy donors and wild-type HCMV-inf ected endothelial cells or fibroblasts. After purification, PMN are su itable for preparation of cytospots which can be used for the antigene mia assay. Cytospin preparations containing a predetermined number of in vitro-generated pp65-positive PMN were used to test some of the maj or parameters involved in performing the antigenemia assay. The result s showed or confirmed that (i) formalin fixation followed by permeabil ization is the best fixation procedure developed to date, (ii) the tes t performance levels provided by different pools of pp65-specific mono clonal antibodies mag be significantly different, and (iii) long-term storage (for an unlimited time) is best achieved by keeping fixed slid es at -80 degrees C, whereas short-term storage (for up to 1 month) is best achieved by keeping unfixed slides at room temperature, This fin ding signifies that slides can be shipped all over the world at room t emperature. In conclusion, the newly developed procedure for in vitro generation of pp65-positive PMN will provide the basis for standardiza tion of the HCMV antigenemia assay and development of quality control programs.