Standardization and Optimization of Pre-Analytical Conditions

An accurate assessment of tissue biomarkers is increasingly important as efforts are made towards individualized molecular targeted therapy of cancer patients. The field of biospecimen science has recognized the impact of biospecimen handling and preanalytical variables on the expression of biomarkers and the need for standardization in biobanking and standard operating procedures (Figure 1). Preanalytical procedures affecting tissue quality are not generally standardized and have been historically poorly controlled. It has been shown that warm and cold ischemic time affect gene and protein expression patterns in the tissue. Many other pre-analytic variables have also been identified, including the size of the tissue, type of fixative used, time of fixation, temperatures during fixation processes, types of tissue processing and paraffin embedding, variations in antigen retrieval and staining protocols, as well as the use of different antibody clones. To address the issue of pre-analytic variation in the clinical setting, physician’s societies published guidelines and white papers. Although the authors of the guidelines attempted to have each parameter supported by scientific evidence, quantitative data are sparse or nonexistent for many variables.

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Figure 1: Current workflow in the pathology laboratory

HistoGeneX is optimizing innovations in tissue processing methods (the pre-analytical phase). We have developed an intelligent vial system (iVial) to control and report pre-analytical variables (EP1913360, US8691165). We have also developed optimal fixation and tissue-processing methods to extract high molecular weight RNA/DNA from formalin fixed paraffin embedded tissue (FFPE) blocks (EP1913359, US8703440).

The iVial system consists of a pre-filled fixative plastic container with a mounted datalogger and a compatible tissue cassette (Figure 2). This is used in the pathology lab during grossing of the surgery specimen where it will automatically start recording time and temperature of fixation from the moment the specimen is inserted into the vial lid (Figure 6).

 

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Figure 2: The pre-filled iVial container with mounted datalogger and tissue cassette

Besides this container with mounted datalogger, there is a second datalogger (Maxim Integrated™ Thermocron® iButton®) that will record time and temperature upon manual activation in the surgery room (Figure 3). The iButton® can be attached to the containers used for transportation of the specimen to the pathology laboratory. The iButton® can then be attached to the iVial to continue monitoring the rest of the process until read-out.

 

 

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Figure 3: The Maxim Integrated™ Thermocron® iButton® and its activator, the Embedded Data Systems™ TC RELAUNCH®

The read-out of the data from these two dataloggers happens through the reading device (Figure 4). This is connected to a PC with installed software to analyze the data (Figure 5).

 

 

 

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Figure 4: The iVial reader exports the data from both dataloggers to a connected pc.

 

 

 

 

 

 

 

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Figure 5: A typical plot showing the measured data during the pre-analytical phase (from excision to start processing) of a tissue specimen

 

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Figure 6: A three-step process to monitor pre-analytical tissue processing conditions

Intellectual Property

Currently, HistoGeneX protects its innovations in Europe and the United States with three granted patents. The iVial patent was awarded by the USPTO and the EPO last year. Our iVial patent has been published in the following European countries: Austria, Belgium, Switzerland, Germany, France, the United Kingdom, Italy and the Netherlands. Besides protection for our iVial, also protection for our fixative solution has been sought. The USPTO has published last year the patent and the EPO is expected to follow.

Potential

These innovations have tremendous potential when combined with existing protocols and machines. That is why we welcome collaborations with companies who can deploy our innovations.

References

1. Mehta S, Shelling a., Muthukaruppan a., et al. Predictive and prognostic molecular markers for cancer medicine. Ther. Adv. Med. Oncol. 2010;2(2):125–148.

2. Vandamme T, Deschoolmeester V, Pauwels P, Peeters M. Tumour markers in metastatic colorectal cancer: clinical implications for treatment with targeted therapy. Belgian J. Med. Oncol. 2012;6:52–57.

3. Neumeister VM, Anagnostou V, Siddiqui S, et al. Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues. J. Natl. Cancer Inst. 2012;104(23):1815–24.

4. Marchiò C, Dowsett M, Reis-Filho JS. Revisiting the technical validation of tumour biomarker assays: how to open a Pandora’s box. BMC Med. 2011;9(1):41.

5. Juhl H. Preanalytical aspects: a neglected issue. Scand. J. Clin. Lab. Invest. Suppl. 2010;242(Suppl 242):63–5.

6. Bai Y, Tolles J, Cheng H, et al. Quantitative assessment shows loss of antigenic epitopes as a function of pre-analytic variables. Lab. Invest. 2011;91(8):1253–61.

7. Wolff AC, Hammond MEH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J. Clin. Oncol. 2007;25(1):118–45.

8. Hammond MEH, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch. Pathol. Lab. Med. 2010;134(7):e48–