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. 2014 Apr;94(4):467-74.
doi: 10.1038/labinvest.2014.7. Epub 2014 Feb 17.

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue

Veronique M Neumeister  1 , Fabio Parisi  1 , Allison M England  1 , Summar Siddiqui  1 , Valsamo Anagnostou  1 , Elizabeth Zarrella  1 , Maria Vassilakopolou  1 , Yalai Bai  1 , Sasha Saylor  1 , Anna Sapino  2 , Yuval Kluger  3 , David G Hicks  4 , Gianni Bussolati  2 , Stephanie Kwei  5 , David L Rimm  1
Affiliations

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue

Veronique M Neumeister et al. Lab Invest. 2014 Apr.

Abstract

While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
A) The performance of 6 marker combinations on the testing and validation subgroup of the Time to Fixation Breast Cancer Series as measured by ROC curves and AUC values. The TQI was then calculated on the complete Time to Fixation Breast Cancer Series. Panel B illustrates the TQI values of Cytokeratin:pHSP27 and Panel C pERK1/2:pHSP27 in relationship with increasing cold ischemic time.
Figure 1
Figure 1
A) The performance of 6 marker combinations on the testing and validation subgroup of the Time to Fixation Breast Cancer Series as measured by ROC curves and AUC values. The TQI was then calculated on the complete Time to Fixation Breast Cancer Series. Panel B illustrates the TQI values of Cytokeratin:pHSP27 and Panel C pERK1/2:pHSP27 in relationship with increasing cold ischemic time.
Figure 1
Figure 1
A) The performance of 6 marker combinations on the testing and validation subgroup of the Time to Fixation Breast Cancer Series as measured by ROC curves and AUC values. The TQI was then calculated on the complete Time to Fixation Breast Cancer Series. Panel B illustrates the TQI values of Cytokeratin:pHSP27 and Panel C pERK1/2:pHSP27 in relationship with increasing cold ischemic time.
Figure 2
Figure 2
Validation and performance of the TQI on the TFBC, the NBT and IBC series: Panel A: Linear regressions between increasing time to fixation (log2 transformed) and the differences of AQUA scores of the TQI markers as performance measurement on an independent built of the TFBC. Higher values correspond with shorter time to fixation. The dotted line shows the 95% CI of the regression line. Panel B: TQI pairs on each time-point of the NBT series. Panel C: Chi squared analysis of the different TQI components. While each marker combination by itself shows a significant correlation to increasing cold ischemic time, the combination of the separate TQI components facilitates the identification of a larger number of samples, which may have compromised tissue quality. Panel D: The TQI performance on the IBC series where special fixation conditions appears to result in significantly less epitope degradation.
Figure 2
Figure 2
Validation and performance of the TQI on the TFBC, the NBT and IBC series: Panel A: Linear regressions between increasing time to fixation (log2 transformed) and the differences of AQUA scores of the TQI markers as performance measurement on an independent built of the TFBC. Higher values correspond with shorter time to fixation. The dotted line shows the 95% CI of the regression line. Panel B: TQI pairs on each time-point of the NBT series. Panel C: Chi squared analysis of the different TQI components. While each marker combination by itself shows a significant correlation to increasing cold ischemic time, the combination of the separate TQI components facilitates the identification of a larger number of samples, which may have compromised tissue quality. Panel D: The TQI performance on the IBC series where special fixation conditions appears to result in significantly less epitope degradation.
Figure 2
Figure 2
Validation and performance of the TQI on the TFBC, the NBT and IBC series: Panel A: Linear regressions between increasing time to fixation (log2 transformed) and the differences of AQUA scores of the TQI markers as performance measurement on an independent built of the TFBC. Higher values correspond with shorter time to fixation. The dotted line shows the 95% CI of the regression line. Panel B: TQI pairs on each time-point of the NBT series. Panel C: Chi squared analysis of the different TQI components. While each marker combination by itself shows a significant correlation to increasing cold ischemic time, the combination of the separate TQI components facilitates the identification of a larger number of samples, which may have compromised tissue quality. Panel D: The TQI performance on the IBC series where special fixation conditions appears to result in significantly less epitope degradation.
Figure 2
Figure 2
Validation and performance of the TQI on the TFBC, the NBT and IBC series: Panel A: Linear regressions between increasing time to fixation (log2 transformed) and the differences of AQUA scores of the TQI markers as performance measurement on an independent built of the TFBC. Higher values correspond with shorter time to fixation. The dotted line shows the 95% CI of the regression line. Panel B: TQI pairs on each time-point of the NBT series. Panel C: Chi squared analysis of the different TQI components. While each marker combination by itself shows a significant correlation to increasing cold ischemic time, the combination of the separate TQI components facilitates the identification of a larger number of samples, which may have compromised tissue quality. Panel D: The TQI performance on the IBC series where special fixation conditions appears to result in significantly less epitope degradation.
Figure 3
Figure 3
Measurement of the TQI performance as a function of ER expression levels quantified by AQUA. Negative TQI values are significantly associated with lower ER AQUA scores on the NBT series and the IBC series (A and B), while the correlation between TQI values and ER expression does not reach significance on build 2 of the TFBC series.
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