[Isolation of cytomegalovirus from peripheral blood by shell-vial culture. Technical considerations]

We performed an study of the possible benefits derived from the use of two vials, incubated for 24 and 72 h respectively, in the isolation of CMV from the leukocyte polymorphonuclear (LPMN) of peripheral blood (viremia) in immunosuppressed patients, using the shell-vial culture technique.

The blood samples with EDTA were fractionated by sedimentation with saline dextran. The LPMN-rich upper layer was used for the isolation of CMV in the shell-vial cell culture (MRC-5 cell line) and to prepare the antigenemia pp65 assay. The cultures were stained at 18-24 h (first vial) and 72 h (second vial) with an indirect immunofluorescence assay with a monoclonal antibody against p72 CMV antigen.

We studied 878 blood samples of which 247 (28.1%) were positive for CMV. Of the positive samples, 211 (85.4%) were detected in the first vial, and 220 (89%) in the second vial. Neither of the two vials detected all positive samples. An overall toxicity of 3%, in the positive samples, and 8.5% in positive samples was detected, respectively. Of the 28 toxic samples, 25.9% were detected in the first vial and 85.1% in the second (p < 0.001). The use of the second vial increased the number of positive samples in 14.5% (36 blood samples).

In view of the results obtained in this study it would seem that, in order to obtain the maximum diagnostic yield in the isolation of CMV from LPMN of peripheral blood in immunosuppressed patients by means of the shell-vial culture, it is necessary to inoculate all the LPMN extracted into two vials. The lengthening of the incubation period up to 72 h for one of the vials leads to an increased in the isolation of CMV of 14.5% with a low overall toxicity.