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EVALUATION OF DIAGNOSTIC HYBRIDS NEW D3 VARICELLA-ZOSTER VIRUS MONOCLONAL ANTIBODY STAIN Pauline Horton, Kathy Erickson, Ana Alvarez, Cindy Powell, Robin Williams, and Mark Neuman, Infectious Diseases & Molecular Diagnostics Division, DSI Laboratories, NCH Healthcare System, Fort Myers, FL Abstract Background: Detection of Varicella-zoster virus (VzV) in clinical specimens can be accomplished by either direct antigen staining of patient cells or tissue culture amplification. In both cases, the quality of the test result can be directly linked with the ability of monoclonal antibody to detect expressed viral antigens. There are currently two commercially available VzV stains for both direct viral antigen detection and culture confirmation. A new 15-minute stain has been developed by Diagnostic Hybrids, Inc. (Athens, OH [DHI]) for both these uses. Objective: The goal of this study was to compare the new DHI VzV stain with the Chemicon VzV stain (Chemicon International, Inc., Temecula, CA). The stains were used for direct antigen detection, and pre-cytopathic effect staining of H&V Mix (DHI) centrifuged enhanced cell culture. Design/Methods: A total of 99 specimens received in M4 viral transport were included in the study. Duplicate Cytospin preparations were made for the direct antigen staining. The slides were fixed in acetone, stained and read according to each manufacturer’s package insert. The H&V Mix culture were inoculated with the supernatant from the specimens according to the H&V Mix PI. The cultures were incubated for 48-hours and then fixed, stained and read according to each manufacturer’s package insert. Results: Each specimen was assessed for sufficient intact, stained epithelial cells before being included in the study data. A total of 59 specimens (60%) had sufficient cells to be included. Direct Smear (N=59) Culture Confirmation (N=99)

Conclusions: The D3 VzV monoclonal antibody stain produces excellent results for both direct antigen and pre-CPE culture identification when compared to Chemicon’s reagent. The H&V Mix culture system is also useful for specimens giving inconclusive smear results due to insufficient cells.

Background Detection of Varicella-zoster virus (VzV) in clinical specimens can be accomplished by either direct antigen staining of patient cells or tissue culture amplification. In both cases, the quality of the test result can be directly linked with the ability of monoclonal antibody to detect expressed viral antigens. There are currently two commercially available VzV stains for both direct viral antigen detection and culture confirmation. A new 15minute stain has been developed by Diagnostic Hybrids, Inc. (Athens, OH [DHI]) for both these uses.

Methods DIRECT SPECIMEN 1. Specimens were centrifuged at 2000 rpm for 10 minutes. 2. The supernatant was removed and sav.ed for culture. 3. The cell sediment from the specimens were washed once with 1-mL of PBS. 4. Duplicate Cytospin preparations were made for the direct antigen staining. 5. The slides were fixed in acetone for 5-minutes. 6. Each cell spot was stained with 25-µL of the D3 MPV stain. 7. The slides were incubated for 15-minutes at 35ºC. 8. Each slide was read for the presence of fluorescing cells. H&V CULTURE 1. H&V cluster plates were processed for culture according to the PI. 2. 200-µL of the specimen supernatant was added to 3microwells in H&V cluster plates 3. The inoculated plates was centrifuged for 60-minutes at 700xg. 4. The plates were incubated for 48 hours at 35ºC. 5. The plates were fixed in 80% acetone for 5-minutes. 6. The plate were stained with 200-µL of the D3 VZV stain and incubated for 15-minutes. Results

Direct Specimen

Results

H&V Culture

H&V Culture

Direct Specimen

Conclusion: The D3 VzV monoclonal antibody stain produces excellent results for both direct antigen and pre-CPE culture identification when compared to Chemicon’s reagent. The H&V Mix culture system found 3 additional positives in specimens containing inadequate cells for direct antigen testing. This makes it useful for specimens giving inconclusive smear results due to insufficient cells

Evaluation of D3 Variccella-zoster Virus Monoclonal Antibody Stain  

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