Fixation on Histology

Fluorescent In Situ Hybridization (FISH)


FISH is a technique that uses a fluorescent labeled probe that attaches to a complementary DNA sequence with a high level of specificity to either metaphase or interphase cells. The double helix of DNA is broken apart (denatured) by heat and chemicals and re-formed (hybridized) with the target probe (O’Connor, 2008). The fluorescent probes (molecules that absorb light of a specific wavelength and emit light of a different, typically longer, wavelength [a process known as fluorescence]) can be either DNA or RNA strands. We use DNA (a double helix formed from two complementary strands of nucleotides held together by hydrogen bonds between G-C and A-T base pairs). Duplication of this genetic information occurs by the use of one DNA strand used as a template for formation of a complementary strand. This is necessary in order for visualization under a fluorescent microscope.

FISH is used to detect chromosomal abnormalities including deletions, duplications and translocations; it is an important test which provides information for the pathology diagnosis and prognosis of various diseases. Our histology department was given the charge to develop a FISH lab based on the state rules and regulations for clinical labs in Florida, which specifically denotes that a Histotechnologist handle paraffin–embedded tissue specimens. We started out by developing the complementary Her2 FISH probe test so we could decrease the turn-around time for reporting cases that needed to have additional testing if they are diagnosed as an Immunohistochemistry (IHC) HER2 positive with a score of 2+. The HER2 (IHC) test, if diagnosed as a 2+ by the American Society of Clinical Oncology (ASCO) guidelines, is required to also have FISH HER2 testing done on the specimen as well as Her2 testing by IHC. In our FISH lab, we developed the FISH HER2 test with a FISH kit approved by the Federal Drug Agency (FDA) specifically for Her2 testing.

In developing the lab, the first thing we needed to accomplish was ensuring the personnel for the FISH lab were all Florida licensed in Molecular Biology to be compliant with the rules and regulations. The current exam is given by ASAP/BOC. We learned denaturing, hybridization, stringency, cross-hybridization, the role of time and temperature, washing of unbound signal, signal intensity and pattern, the effect of different types of immersion oil, trouble shooting, and quality requirements.

The next step was test validation. This is to determine the probe’s specificity, sensitivity, accuracy, and reproducibility. With consultation of a pathologist, we established clearly defined scoring criteria for determining that the assay was acceptable. This included equivocal cases and those cases where the techs were not in concordance. The test validation also confirms the cut-off and the interpretation of the test to meet College of American Pathologists (CAP)/ASCO Guidelines. We originally started the validation using the CAP/ASCO Guidelines from 2007 that were later updated in 2013; now we adhere to the most current 2018 CAP/ASCO Guidelines (Wolff et al., 2018). In establishing our test parameters we recruited a certified medical technologist to complete the specificity part of the validation. The technologist needed to be certified in reading karyotypes in metaphase cells. Metaphase scoring is still considered to be the “gold standard” for establishing the expected signal pattern at the correct chromosomal location (Gu, Smith, & Dowling, 2017).

One of the most challenging tasks we faced was learning how to enumerate or count significant indicators within the assay. Thus far, the manual counting of signals per cell is still the standard approach, making data analysis the post-experimental rate-limiting step. The diagnostic decisions are often based on the ratio of the number of signals from two distinct chromosomal regions to determine chromosomal or gene amplification (Huber, Voith von Voithenberg, & Kaigala, 2018). Our technologists traveled to Abbott Laboratories in Chicago, Illinois for training. The training topics covered the technical background of FISH, the processing of the tissue samples, and the enumerations of the samples using the fluorescent microscope (Abbott Laboratories, 2010). A comparison study was conducted which encompassed forty positive and forty negative tissue samples for two Histotechnologists to enumerate. Results were compared with other laboratories offering the test after reviewing parallel samples and investigation of any discrepant results. The Histotechnologists had to distinguish different signal representation and acquire the skills to determine tumor vs. non-tumor and in-situ vs. invasive. They also had to learn how to accurately determine the number of signals in each cell (Wolff et al April 2007). The ratio of target (red) compared to the control (green) in a total of 60 cells, in addition to the average copy number of Her2 signals per cell, were used to determine positive or negative test results (Gu et al., 2017).

The report had several requirements set forth by the CAP including time fixed in formalin for breast tissue samples, kit and vendor selection, information on how the test was validated including FDA/FDA Modified or Lab Developed Tests, staff training, operating procedures, forms, checklists, and report building. These tasks needed to be taken care of before the test could be implemented (Gu et al., 2017). The workflow related to how the results were going to be reviewed by the pathologist, signed out, and reported also became a challenge.

Once the test was up and running, we had to address CLIA regulations which require review of the performance characteristics of each FISH test to assess for problems related to the assay. We established a resolution to this requirement by securing a CAP proficiency biannual exam for FISH Her2. The proficiency test is named “CYH” and is used to verify that staff is proficient in enumeration of the samples. There are ten tissue samples provided by CAP. The lab is required to probe and enumerate each sample and pass with a 90% concordance.

We are moving forward to incorporate a wider span of FISH testing and, as we grow, we have also come to the realization that we need an experienced molecular PhD to lead us into the future. We are excited about the new FISH tests we currently have in the pipeline and we are looking forward to expanding our testing abilities to better aid in the diagnosis of our patients.


Her2 Negative Case


Her2 Positive Case

Written by Nancy Shellhorn, HT(ASCP), Tracy Majewicz, MB(ASCP), HTL(ASCP), Fayrouz Manadilo, MB (ASCP), HTL(ASCP)


  • Abbott Laboratories. (2010). PathVysion, the HER2 test for women with breast cancer. Customer Training Guide List No. 02N18-02.

  • Gu, J., Smith, J. L. & Dowling, P. K. (2017). Fluorescence in situ hybridization probe validation for clinical use. Cancer Cytogenetics: Methods and Protocols, Methods in Molecular Biology, 1541. DOI 10.1007/978-1-4939-6703-2_10.

  • O’Connor, C. (2008). Fluorescence in situ hybridization (FISH). Chromosomes and Cytogenetics.

  • Huber, D., Voith von Voithenberg, L., & Kaigala, G. V. (2018). Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH? Micro and Nano Engineering, 1, 15-24.

  • Wolff, Antonio C., Hammond, M. Elizabeth Hale, Allison, Kimberly H., Harvey, Brittany E., Mangu, Pamela B. (2018). Human epidermal growth factor receptor 2 testing in breast cancer. Archives of Pathology & Laboratory Medicine.

  • Wolff, D. J., Bagg, A., Cooley, L. D., Dewald, G. W., Hirsch, B. A., & Jacky, P. B. (2007). Guidance for fluorescence in situ hybridization testing in hematologic disorders. Journal of Molecular Diagnostics, 9(2).