The Biospecimen Research Database

The purpose of this study was to assess the potential effects introduced by delay to fixation (DTF) on immunohistochemical staining of cancer biomarkers in prospectively collected FFPE tumor specimens under the National Cancer Institute's Pre-analytical Variable (BPV) program.  Tumors from 111 kidney, 29 colon, 28 ovarian, and 8 lung resections were collected at four medical institutions using a common set of standard operating procedures (SOPs). Each surgical resection was dissected to yield tumor segments of approximately equivalent size (0.33 cm x 0.5 cm)  which were stored for 1 h (control), 2 h, 3 h, or 12 h in a humidified chamber at room temperature before fixation.  Tumor segments were then fixed in 10% neutral buffered formalin (NBF) at room temperature for 10-12 h before paraffin-embedding.  Depending on the tissue, between 5 and 13 biomarkers were examined in FFPE sections (5 µm) by immunohistochemistry.  Positive immunostaining was quantified using Aperio software after calibration using slides manually scored by a pathologist.  H-score was calculated by multiplying the percentage of positively stained cells/nuclei by the intensity of the stain (0-3). Statistical analysis included the mean and percent difference in H-scores for experimental (2, 3, 12 h DTF) specimens relative to corresponding 1 h DTF controls.  H-score data collected from BPV tumor specimens was used to calculate the estimated probability of a change in intensity score (0-3) for each DTF timepoint relative to the 1 h DTF control.  Statistical significance was set to p<0.05. A subset of specimens stored as FFPE blocks were re-evaluated one year post-collection.

The purpose of this study was to assess the potential effects associated with time in fixative (TIF) on immunohistochemical staining of cancer biomarkers in prospectively collected FFPE kidney tumor specimens under the National Cancer Institute's Pre-analytical Variable (BPV) program.  Tumors from 60 kidney resections were collected at four medical institutions using a common set of standard operating procedures (SOPs). Each surgical resection was dissected to yield tumor segments of approximately equivalent size (0.33 cm x 0.5 cm) that included a quality control for histopathology, a snap-frozen control, and four experimental tumor segments.  After a DTF of 1 h, experimental segments were placed in 10% NBF at room temperature for 6, 12 (control), 23, or 72 h. A total of 13 biomarkers were examined in FFPE sections (5 µm) by immunohistochemistry.   Positive immunostaining was quantified using Aperio software after calibration using slides manually scored by a pathologist.  H-score was calculated by multiplying the percentage of positively stained cells/nuclei by the intensity of the stain (0-3). Statistical analysis included the mean and percent difference in H-scores of experimental (6, 23, 72 h TIF) specimens relative to corresponding 12 h TIF controls. H-score data collected from BPV tumor specimens was used to calculate the estimated probability of a change in intensity score for each TIF timepoint relative to the 12 h TIF control.  Statistical significance was set to p<0.05. A subset of specimens stored as FFPE blocks were re-evaluated one year post-collection for RCC1 immunostaining.