Changes

6.6.2.5 Disaggregation methods should be optimized for different tissue types:

6.6.2.5 Disaggregation methods should be optimized for different tissue types:

    a. Disaggregation of solid tumor samples for tissue culture is needed. Mechanical and/or enzymatic methods may be used. If sufficient tumor material is submitted, both methods of disaggregation are recommended. For some tumor types, different growth characteristics can be seen with exposure to collagenase versus no exposure to collagenase. If sufficient material is available, cultures should be initiated with and without enzyme exposure.

::a. Disaggregation of solid tumor samples for tissue culture is needed. Mechanical and/or enzymatic methods may be used. If sufficient tumor material is submitted, both methods of disaggregation are recommended. For some tumor types, different growth characteristics can be seen with exposure to collagenase versus no exposure to collagenase. If sufficient material is available, cultures should be initiated with and without enzyme exposure.

    b. Disaggregation of lymphoid tissues into single cell suspension is necessary before culture initiation. The lymphoid cells in most tissues are readily disaggregated by mechanical means such as mincing with scalpels or curved scissors. The use of these methods is often advantageous if the tissue is easily dissociated because it will keep the loss of cells to a minimum and may help minimize stromal contamination because stromal cells are often locked in fibrous connective tissues. If cells are not readily liberated by mechanical means, enzymatic digestion may be necessary. When using enzymatic digestion, the tissue must first be minced and then incubated with the enzyme solution (e.g., collagenase) for 20 minutes to 16 hours depending on how quickly cell release occurs.

::b. Disaggregation of lymphoid tissues into single cell suspension is necessary before culture initiation. The lymphoid cells in most tissues are readily disaggregated by mechanical means such as mincing with scalpels or curved scissors. The use of these methods is often advantageous if the tissue is easily dissociated because it will keep the loss of cells to a minimum and may help minimize stromal contamination because stromal cells are often locked in fibrous connective tissues. If cells are not readily liberated by mechanical means, enzymatic digestion may be necessary. When using enzymatic digestion, the tissue must first be minced and then incubated with the enzyme solution (e.g., collagenase) for 20 minutes to 16 hours depending on how quickly cell release occurs.

6.6.2.6 Culture methods, culture medium, and culture conditions should be chosen to best support the type of tumor received.

6.6.2.6 Culture methods, culture medium, and culture conditions should be chosen to best support the type of tumor received.

    a. The diagnosis and histopathology of a tumor can be helpful in determining culture and harvest methods. Different cell types can be expected to respond differently with growth medium, harvest method, and other factors (Table 6). If the diagnosis is unknown at culture initiation, it can be helpful to know whether the pathologist would classify the tumor as a “small round cell tumor” (SRCT), which includes lymphoproliferative disorders. SRCTs can be successfully grown in suspension, whereas non-SRCTs are best grown with monolayer (flask or coverslip) culture methods. Most, but not all, SRCTs (e.g., lymphoproliferative disorders) will also grow in monolayer culture. If adequate tissue is obtained, both culture types should be initiated for SRCTs. For very small tumor samples, coverslip cultures are recommended. Duplicate cultures should be established whenever possible.

::a. The diagnosis and histopathology of a tumor can be helpful in determining culture and harvest methods. Different cell types can be expected to respond differently with growth medium, harvest method, and other factors (Table 6). If the diagnosis is unknown at culture initiation, it can be helpful to know whether the pathologist would classify the tumor as a “small round cell tumor” (SRCT), which includes lymphoproliferative disorders. SRCTs can be successfully grown in suspension, whereas non-SRCTs are best grown with monolayer (flask or coverslip) culture methods. Most, but not all, SRCTs (e.g., lymphoproliferative disorders) will also grow in monolayer culture. If adequate tissue is obtained, both culture types should be initiated for SRCTs. For very small tumor samples, coverslip cultures are recommended. Duplicate cultures should be established whenever possible.

    b. For lymphoid tissues, disaggregated cells are cultured in suspension using appropriate supportive growth medium. Tumor cells are spontaneously dividing; however, mitogens may be used for lymphoid disorders to encourage proliferation of the desired cell type.

::b. For lymphoid tissues, disaggregated cells are cultured in suspension using appropriate supportive growth medium. Tumor cells are spontaneously dividing; however, mitogens may be used for lymphoid disorders to encourage proliferation of the desired cell type.

6.6.2.7 Experience with solid tumor culture will provide the laboratory with information regarding optimal growth conditions and harvest methods for different tumor types.

6.6.2.7 Experience with solid tumor culture will provide the laboratory with information regarding optimal growth conditions and harvest methods for different tumor types.

    a. It can be helpful for the laboratory to maintain a database that documents how the different tumor types have grown and which culture and harvest conditions yield abnormal clones. This database can then be searched for optimal processing and harvesting methods for any new tumor received in the laboratory.

::a. It can be helpful for the laboratory to maintain a database that documents how the different tumor types have grown and which culture and harvest conditions yield abnormal clones. This database can then be searched for optimal processing and harvesting methods for any new tumor received in the laboratory.

    b. Short culture durations are preferred to optimize the mitotic index of early dividing tumor cells and to avoid growth of normal tissues. Depending on the amount of available tissue, a combination of direct, 24-hour, and/or 48-hour cultures are most often utilized for lymphoid disorders. Short-term cultures (e.g., direct or overnight cultures) may also be used in conjunction with longer-term cultures to capture actively dividing cells from solid tumors.

::b. Short culture durations are preferred to optimize the mitotic index of early dividing tumor cells and to avoid growth of normal tissues. Depending on the amount of available tissue, a combination of direct, 24-hour, and/or 48-hour cultures are most often utilized for lymphoid disorders. Short-term cultures (e.g., direct or overnight cultures) may also be used in conjunction with longer-term cultures to capture actively dividing cells from solid tumors.

    c. Frequent (daily) observation of cells in culture is needed to determine cell growth rate and optimal time to harvest. Tumor cells should be harvested as soon as possible upon adequate growth to capture early dividing tumor cells and to prevent overgrowth by chromosomally normal cells.

::c. Frequent (daily) observation of cells in culture is needed to determine cell growth rate and optimal time to harvest. Tumor cells should be harvested as soon as possible upon adequate growth to capture early dividing tumor cells and to prevent overgrowth by chromosomally normal cells.

    d. Conditions used for cell harvest will vary among tissue types (e.g., mitotic inhibitors) used (e.g., colcemid, velban, ethidium bromide), their concentration, and exposure duration, and they should be established by each laboratory.

::d. Conditions used for cell harvest will vary among tissue types (e.g., mitotic inhibitors) used (e.g., colcemid, velban, ethidium bromide), their concentration, and exposure duration, and they should be established by each laboratory.

== Analytical Methods ==

== Analytical Methods ==