The tests for detection of cytopathogenic and/or hemadsorbing agents provided in this section shall be conducted when prescribed in an applicable Standard Requirement or in the filed Outline of Production for a product.

(a) Test for cytopathogenic agents. One or more monolayers that are at least 6 cm2 and at least 7 days from the last subculture shall be tested as provided in this paragraph.

(1) Stain each monolayer with a suitable cytological stain.

(2) Examine the entire area of each stained monolayer for evidence of inclusion bodies, abnormal number of giant cells, or other cytopathology indicative of cell abnormalities attributable to an extraneous agent.

(b) Test for hemadsorbing agents. One or more monolayers that are at least 6 cm2 and at least 7 days from the last subculture shall be tested as provided in this paragraph.

(1) Wash the monolayer with several changes of phosphate buffered saline.

(2) Add an appropriate volume of a 0.2 percent red blood cell suspension to uniformly cover the surface of the monolayer of cultured cells. Suspensions of washed guinea pig and chicken red blood cells shall be used. These suspensions may be mixed before addition to the monolayer or they may be added separately to individual monolayers.

(3) Incubate the monolayer at 4 °C for 30 minutes, wash with phosphate buffered saline, and examine for hemadsorption.

(4) If no hemadsorption is apparent, repeat step (b)(2) of this section and incubate the monolayers at 20-25 °C for 30 minutes, wash with phosphate buffered saline, and examine again for hemadsorption. If desired, separate monolayers may be used for each incubation temperature.

(c) If specific cytopathology or hemadsorption attributable to an extraneous agent is found, the material under test is unsatisfactory and shall not be used to prepare biological products. If an extraneous agent is suspected because of cytopathology or hemadsorption and cannot be eliminated as a possibility by additional testing, the material under test is unsatisfactory.

[50 FR 441, Jan. 4, 1985, as amended at 58 FR 50252, Sept. 27, 1993]