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Journal of Clinical Microbiology, December 1998, p. 3585-3589, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Standardization of the Human Cytomegalovirus Antigenemia Assay by Means of In Vitro-Generated pp65-Positive Peripheral Blood Polymorphonuclear Leukocytes

Giuseppe Gerna,* Elena Percivalle, Maria Torsellini, and M. Grazia Revello

Servizio di Virologia, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, 27100 Pavia, Italy

Received 19 June 1998/Returned for modification 13 August 1998/Accepted 17 September 1998

We generated in vitro human cytomegalovirus (HCMV) pp65-positive polymorphonuclear leukocytes (PMN) resembling those detected in vivo, following cocultivation of PMN from healthy donors and wild-type HCMV-infected endothelial cells or fibroblasts. After purification, PMN are suitable for preparation of cytospots which can be used for the antigenemia assay. Cytospin preparations containing a predetermined number of in vitro-generated pp65-positive PMN were used to test some of the major parameters involved in performing the antigenemia assay. The results showed or confirmed that (i) formalin fixation followed by permeabilization is the best fixation procedure developed to date, (ii) the test performance levels provided by different pools of pp65-specific monoclonal antibodies may be significantly different, and (iii) long-term storage (for an unlimited time) is best achieved by keeping fixed slides at -80°C, whereas short-term storage (for up to 1 month) is best achieved by keeping unfixed slides at room temperature. This finding signifies that slides can be shipped all over the world at room temperature. In conclusion, the newly developed procedure for in vitro generation of pp65-positive PMN will provide the basis for standardization of the HCMV antigenemia assay and development of quality control programs.

* Corresponding author. Mailing address: Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy. Phone: 39-382-502644/34. Fax: 39-382-502599. E-mail: Virology{at}ipv36.unipv.it.

Journal of Clinical Microbiology, December 1998, p. 3585-3589, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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