Expanding the Role of Cytotechnologists in Molecular Diagnostics

Expanding the Role of Cytotechnologists in
Molecular Diagnostics

Jesse S. Voss, MB, CT(ASCP)
Mayo Clinic
Rochester, Minnesota

Molecular diagnostics is a rapidly expanding field, and there is a need for highly skilled technologists to perform genetic testing. One aspect that makes cytotechnologists (CTs) unique when compared with other technologists is the ability to combine existing morphology skills with molecular techniques. CTs are well suited to perform testing due to the extensive training and understanding of neoplasia and other diseases as well as intensive morphologic training, which may be more difficult to teach “on the job” to other technologists. Genetics and molecular techniques can be effectively taught to supplement these existing skill sets. Over the past decade, we have progressively integrated CTs in different areas of molecular and other ancillary techniques, some of which include fluorescence in situ hybridization (FISH) analysis, circulating tumor cell (CTC) detection, prescreening for acid fast bacilli (AFB) and quantitative immunohistochemical (IHC) stained slides and tumor identification for molecular genetic testing.1

FISH Analysis

Figure 1: Click image above
for full image and description

Mayo Clinic and many other institutions currently use cytotechnologists to perform a wide variety of molecular testing. The most common area of molecular testing performed by CTs is FISH. FISH testing is currently performed on cytology specimens including urine, biliary tract brushings, bronchial brushings and Barrett’s esophageal brushings. CTs are very well suited to perform cytology-based FISH tests since they incorporate morphologic assessment and locator skills already used in routine cytology analyses. Screening a FISH slide is very similar to screening a routine cytology Pap-stained slide. FISH slides are screened in a systematic fashion, ensuring adequate overlap of each field of view while looking for morphologically abnormal cells. Much like routine cytology, CTs are very good at recognizing differences at the cellular level using FISH. Morphology skills are used to delineate differences in nuclear size, chromatin texture seen with DAPI, architecture of cell groups and differentiating epithelial and inflammatory cells. We also must be able to correctly interpret the signal pattern from these cells and discriminate true signals from artifacts.

FISH interpretation can be similar to considering differential diagnoses in urine cytology. When we find an atypical-appearing urothelial cell during routine cytology, we consider numerous entities such as a reactive/reparative mechanism, degeneration, radiation changes, polyomavirus or cancer. We use the same concept when evaluating an abnormal appearing cell by FISH. Other artifacts or entities that mimic a polysomic cell must be considered, some of which include overlapping cells, signal splitting and non-specific signals (Figure 1).

CTC Analysis

Figure 2: Click image above
for full image and description

Another area that has received a lot of attention lately is the detection of CTCs in patients with a metastatic disease, particularly in those with metastatic breast, colon or prostate cancer. Numerous highly sensitive technologies have been developed during the last few years to detect as little as a few CTCs in one mL of whole blood. While many studies have reported the prognostic value of CTC detection, there is only one FDA-approved platform. The Cellsearch® CTC (Veridex, Raritan, NJ) test has shown to provide prognostic information in patients with metastatic breast, prostate or colon cancer. This assay uses a cell harvest instrument to extract CTCs out of a blood sample by using magnetic beads coated with antibodies to the epithelial marker EPCAM. Cells are then labeled with CD45, cytokeratins (CK) 8, 18 and 19 and DAPI, which are used to distinguish CTCs from white blood cells and non-specific debris. The cells are placed in a cartridge and scanned by an automated fluorescence microscope, which takes pictures of the captured cells.

The cytotechnologists are also well suited to perform Cellsearch CTC analysis using their existing morphologic skills. CTs will prescreen a gallery of images and highlight the CTCs and suspicious looking cells that are then reviewed by a pathologist (Figure 2). The cells are displayed in a gallery where each cell is shown in a row. The first column is a composite of CK staining (shown in second column) that stains the CTCs and DAPI staining, which is a nuclear counterstain (shown in third column). The fourth column displays the CD45 staining, which is a marker for white blood cells and the last column on the right is the FITC channel that shows any non-specific signal used to identify debris and other artifacts. The CTs will scan for CTCs that display a combination of CK and DAPI staining but without CD45 staining, while ruling out any artifacts that may be present. Some of these artifacts include non-specific junk, fuzzy signals, unmatched DAPI/CK signals, and on very rare occasions, positive control cell carryover (Figure 2). 

IHC and AFB Slide Analysis

Cytotechnologists at our institution are also used routinely to prescreen slides stained for AFB and to quantitate HER2, Ki-67 (MIB1), estrogen receptor (ER) and progesterone receptor (PR) immunohistochemical (IHC) stained slides. The CTs will organize the cases, resolve any discrepancies (fixation time, specimen source, etc), prescreen the slides, mark in situ and invasive areas of tumor, prepare paperwork and enter preliminary diagnoses. All results are then reviewed by a pathologist. We do not see this as replacing a pathologist, but as assisting them, similar to prescreening non-gyn slides as done in routine cytology. We have found that all of these activities help save pathologist time and increase overall efficiency. Analyses can be performed using manual (semi-)quantitation or with digital image analysis assistance.

We performed an internal validation study showing that CT image analysis of HER2 stained breast cancer slides with a brief pathologist review provided a lower equivocal (2+) rate with a higher percentage of these cases being FISH positive than consensus pathologist manual assessments.2 We also performed a reproducibility study comparing the intraobserver and interobserver reproducibility rates of three pathologists using manual assessments, three CTs using manual assessments and the same three CTs using image analysis assistance with the ACIS instrument.3 This study showed that when compared to FISH, the CT ACIS-assisted method had a higher concordance than CT or pathologist manual assessments. In addition, average kappa scores (measuring concordances) were highest using CT ACIS assistance when evaluating intraobserver and interobserver reproducibility. We feel that these data demonstrate that CTs can effectively perform preliminary IHC analysis during routine clinical practice and support the use of CTs in these situations.

Tumor Identification and Molecular Genetic Testing

Figure 3: Click image above
for full image and description

One of the fastest growing areas of molecular testing includes mutation testing of paraffin embedded tumor tissue specimens to determine specialized pathway inhibitor drug therapy in cancer patients. Common tests include KRAS, BRAF and EGFR gene mutation testing for colon cancer, as well as for lung cancer or melanoma; and numerous applications are being discovered at a rapid pace. Mutation testing on cytology samples, typically FNA samples and other easily obtainable specimens, will likely increase in frequency during the coming years. This will give rise to opportunities of incorporate molecular testing and morphology interpretations in routinely obtained cytology samples. While this is a new and exciting area for the cytology lab, some barriers do exist. CTs will be required to learn different skill sets required for PCR testing that may require additional training and education. Another potential problem may include the changing regulatory landscape where CTs are not allowed to perform molecular testing in certain states.

While CTs are fully capable of performing traditional molecular testing (i.e. PCR, etc), there are other pre-analytical tasks that must be performed prior to analysis. At our institution, CTs perform a preliminary sample review to mark areas of tumor and determine the tumor cell percentage in a particular patient specimen. This is a crucial component of tumor specimen genetic testing since the analytical performance of PCR based tests are dependant on the concentration of tumor cells in the specimen. This has commonly been done by a molecular pathologist in the past, but we have recently taken advantage of CTs morphologic skills to perform this preliminary specimen review. The CTs will dot areas of H&E slides to be scraped from an unstained slide and estimate tumor percentage, which is then briefly reviewed by a pathologist (Figure 3). This concept may also be applied to cytology specimens where a slide would be screened for cancer cells and then scraped off the slide for molecular testing and cytotechnologists would be ideal for this situation. In addition, CTs at our institution have recently been performing a preliminary review of cases undergoing hereditary non-polyposis colorectal cancer testing (HNPCC) and tumor identification has been expanded to marking tumor on tissue slides prior to FISH analysis.

Benefits of CT Utilization

While these CT activities may not be directly attributed to an increase in revenue (i.e., most CT activities discussed cannot be directly billed), we have found that pathologists are increasingly more productive during clinical caseloads and are able to perform more complex testing and other revenue-generating activities. We find that CT analysis of FISH, CTC, IHC, and H&E slides from cases for molecular testing has a utility similar to prescreening GYN and non-GYN slides during routine cytology. The activities performed by CTs during molecular testing allow the pathologist to be more efficient, which can result in cost savings. Using our workload recording data, we were able to demonstrate an increase in pathologist efficiency by using CTs in a supporting role.1 

This analysis revealed the potential savings of utilizing CTs in molecular diagnostics using the average CT and pathologist salaries taken from most recent ASCP and 2009 Modern Healthcare surveys. We noticed a potential 96% decrease in pathologist analysis time, resulting in a 71% overall decrease in salary expense for FISH testing by having CTs evaluate a FISH case with a brief pathologist review over having a pathologist perform the analysis alone. For ER, PR, Ki-67, and HER2 analysis of breast cancers, we noticed an 86% decrease in pathologist analysis time, resulting in a 67% overall decrease in IHC salary expense by having a CT analysis with a brief pathologist review versus having a pathologist review without CT analysis. For Cellsearch CTC analysis, we noticed a 71% decrease in pathologist analysis time resulting in a 41% overall decrease in salary expense for CTC testing by have a CT analysis with a brief pathologist review versus having a pathologist review alone. For CTs review of paraffin-embedded tumors for molecular genetic testing, we noticed a 33% decrease in pathologist analysis time during the first few months of implementation, but the additional CT time offset most of the savings associated with testing support. However, after one year of testing we have been able to streamline this process and further cost savings and increased productivity have been attained. We have found that with time and experience, pathologists become more confident in the cytotechnologists abilities and are able to review cases more quickly, further increasing efficiency. These methods are being revised and improved and other areas of opportunity are being explored to increase efficiency even more. 

Educational Opportunities

While numerous opportunities to expand molecular testing will occur in the near future, additional CT training will be required to effectively perform this testing. There are numerous literature resources and formal training courses that can be useful. One thing that would be beneficial for any technologist interested in performing PCR testing is to obtain the Molecular Biology (MB) certification from the ASCP. All ASCP certified technologists qualify and further information can be obtained on the ASCP Web site. Content guidelines and the reading list include many books and study materials to become more familiar with molecular techniques. There are also formal hands-on training and review courses to prepare for the exam. In addition, the ASC offers an excellent review course that was developed by numerous ASC members. The “Core Curriculum in Molecular Pathology” course is easily accessible on the ASC Web site that is free to all ASC members and can be found through the “Education” and “eLibrary” links. Studying for and taking the MB exam offers a good introduction to molecular testing and can prepare CTs to perform this type of testing.

In conclusion, CTs can be utilized effectively in certain situations for specimen analysis relating to molecular diagnostics to increase pathologist efficiency. Morphology-based applications in molecular testing are best suited for CTs due to their existing skills and knowledge of neoplastic conditions, but additional molecular-based training may be required. There are numerous opportunities to expand the role of CTs to include support activities and testing in molecular and tissue applications.



  1. Root RM, et al. Expanding the role of cytotechnologists: A cost effective approach for anatomic pathology at mayo clinic. Cancer Cytopathol 2010;118 (Suppl S) :359-360 (abstract).
  2. Minot DM, et al. Automated cellular imaging system iii for assessing HER2 status in breast cancer specimens: Development of a standardized scoring method that correlates with FISH. Am J Clin Pathol 2009;132:133-138.
  3. Minot DM, et al. Image analysis of HER2 immunohistochemical (IHC) stain – reproducibility and concordance with fluorescence in situ hybridization (FISH) of a laboratory validated scoring technique. Am J Clin Pathol 2011; (In press).