Managing an Established Cytogenetics Lab

September 2021 - Vol.10 No. 8 - Page #14
Category: Genetic Testing

For the first part of this series on cytogenetics, please visit

As the concept of cytogenetics continues to proliferate throughout medical laboratory and pathology practices, it is important to keep in mind the core basics necessary to open and maintain a cytogenetics laboratory. In the first part of this article series, I discussed the basic tenets of opening a new cytogenetics lab and the focus moving forward is on operations.

As many laboratorians are aware, cytogenetics is essentially the study of the structure of chromosomes and its relationship to disease. What may be less well known is that most samples received in a cytogenetics laboratory are referred for oncology (particularly for hematological diseases), as opposed to constitutional genetic conditions. As another tool in a hospital’s arsenal, cytogenetics operating in conjunction with molecular pathology, hematology, and flow cytometry, provides pathologists, oncologists, and other clinicians with a well-rounded view of a patient’s condition.

Laboratory Specialty Teamwork

To further illustrate the strength of combining knowledge, experience, and technology from multiple disciplines in the lab, consider, for example, how hematology and flow cytometry provide information about the exterior of the cell, while cytogenetics and molecular pathology contribute to the clinicians’ knowledge of the genetics of the cell. These fields can therefore work together to determine if the patient has leukemia, what type of leukemia, the stage of the disease, and the prognosis.

Specific to patients with leukemia, fluorescence in situ hybridization (FISH) determines whether the patient is BCR/ABL1 positive; PCR determines if the patient has CML, ALL, or AML; and cytogenetics determines if the patient only has a t(9;22) translocation or if other cytogenetic abnormalities are present and their potential effect on the patient’s prognosis (see FIGURE 1).

Test Menu Establishment

Now that the impact of cytogenetics is apparent, the next step is to setup the laboratory, which was covered in my previous article. Moving beyond that, an obviously important aspect of early operations is determining the test menu for your institution.

Much of the test menu decision making is determined by reviewing what types of samples are sent out for chromosome testing and what send-out FISH testing is being ordered and then reviewing whether these can be brought in house. In this stage, it is important to have discussions with your oncologists about which specific FISH probes they would like included on a FISH panel. Remember to monitor the capacity of your personnel when making these decisions, as the technologists perform not only the wet lab work, but also the analysis, which can be very time consuming.

Forecast Testing Cost

Once the necessary test menu has been established, the next step is to perform cost analyses for each test. One efficient way to do this is to build a spreadsheet that lists consumables (eg, pipette tips), number of each consumable needed per test, name and number of reagents needed for each test, and per-unit cost per test (see FIGURE 2).

Fortunately, most hospitals have cost analysis templates that include information such as CPT codes, test name, reagents and consumables cost, number of expected tests run per week, days of the week the test is run, number of controls run, estimated repeat percentage, proficiency testing costs, validation costs, labor cost per run (hands on time), and the need for additional staff or equipment (see FIGURE 3).

Validation, PT, QA and QC

After the cost analysis has been approved by administration, begin to map out a validation plan for each test and determine how proficiency testing will be performed. This is also an appropriate time to develop QC metrics and expected ranges and values. Interestingly, most cytogenetics labs are well entrenched with few new ones being established (yet), so the availability of benchmarking information on validating chromosome analysis is limited. Therefore, I am happy to share our approach. We looked at a few key criteria for validating our testing:

  • Set up cultures in a sterile manner
  • Monitor for cell growth
  • Determine miotic index
  • Abnormality detection

These criteria were documented for each sample type that the laboratory was planning on analyzing. Each sample has a recommended culture media and supplements depending on sample type and suspected diagnosis, as well as number of days in culture, recommended number of cultures to set up based on suspected diagnosis (a minimum of two cultures placed in two separate incubators), and number of cells to count, analyze, and karyotype. To help round out your program, the AGT Cytogenetics Laboratory Manual1 is an excellent resource for media and supplement recommendations and has well-defined protocols for chromosome analysis and FISH. Furthermore, The American College of Medical Genetics and Genomics (ACMG) Technical Standards for Clinical Genetics Laboratories Section E2 and the CAP cytogenetics checklist both indicate minimum requirements for incubator conditions, types of cultures, and number of cells to count, analyze, and karyotype.3 The CAP has proficiency testing available for chromosome analysis of multiple specimen types.

FISH Management

There are several informative articles on FISH validation now available and the ACMGG Technical Standards for Clinical Genetics Laboratories (E9) and the CAP checklist list minimum requirements for FISH validation as well.2-6

Keep in mind, for each new probe introduced to the lab, a series of studies is performed including probe familiarization, probe localization, probe sensitivity and specificity, analytical sensitivity and specificity, the development of a normal database for interphase probes (to determine cutoff levels for each signal pattern), as well as a blinded study. These studies vary slightly based on probe design—enumeration, fusion, or break apart—and sample type—metaphase, interphase, stimulated peripheral blood, amniocentesis, bone marrow, or formalin-fixed.

Once a FISH probe has been initially validated, each shipment likewise needs to be validated, typically by processing a sample with probes from both the old and new shipments and comparing the results. The ACMGG and CAP recommend biannual or continuous monitoring of quality for FISH probes, including hybridization efficiency, probe sensitivity, and the presence of background or other technical issues that may impact test interpretation. In addition to FISH validation requirements, the CAP has proficiency tests available for most FISH tests based on probe design or sample type. If a proficiency test is not available from CAP, sample exchanges with other laboratories are a good option for alternative assessments.

Ensure Proper Coding and Billing

In addition to the tasks related to the testing itself, equally important are processes related to test ordering, billing, CPT codes, and creating report templates for your hospital’s reporting system. The CAP has a list of things that need to be included in the patient’s report, including reason for referral, number of cells counted, analyzed, and karyotyped, all using appropriate nomenclature. The International System for Human Cytogenomic Nomenclature (ISCN) contains descriptions and examples of abnormal cytogenetic, FISH, and microarray nomenclature.7 The report should also include the implication for prognosis, the need for genetic counseling, and recommendations for future studies as needed. It is good practice to include a disclaimer describing the limitations of the testing and whether the test is FDA cleared and the laboratory has validated the test appropriately.

Many laboratories include CPT codes in their reports in addition to being part of the billing process. CPT codes for chromosome analysis include a tissue culture code based on specimen type, a chromosome analysis code based on the number of cells counted and karyotyped, and an interpretation code. The following CPT codes are examples of these:8

  • 88237—culturing bone marrow cells
  • 88262—15 to 20 cells, 2 karyotypes
  • 88291—interpretation and report

Likewise, CPT codes for FISH include processing and number of cells analyzed, one for the FISH probe times the number of probes used, and an interpretation code. For example, with a BCR/ABL1 FISH test on 200 interphase cells, the CPT codes would be:8

  • 88275—100 to 300 cells analyzed
  • 88271 x 2—for two FISH probes
  • 88291—interpretation and report

QA and QC Requirements

Concurrent to the development of the validation plan, consider the metrics you plan to monitor for quality assurance and quality control. In addition to tracking any issues with specimens, result discrepancies, and turnaround times (TATs), other metrics specific to a cytogenetics laboratory should be taken into account. As an example, not only do we track culture failures, but we also track insufficient cells for analysis and reasons for a lack of dividing cells (and thus an incomplete study). Further, the CAP indicates minimum TATs and failure rates for different specimen types, as well as specific TATs for STAT chromosome cases. The cytogenetics lab should develop expected detection rates for chromosome abnormalities observed in specific leukemias based on information from the scientific literature.

Specific to FISH, there are similar metrics to track. These include:

  • Insufficient hybridization
  • Changes in hybridization efficiency, probe sensitivity, or signal patterns
  • Expected abnormality rate based on specimen type and disease

All metrics should be reviewed monthly by the laboratory director to detect trends and identify further areas for improvement.


As you learn more about starting or expanding a cytogenetics laboratory, please keep in mind that almost all areas of the laboratory involve manual work. That said, automation in this area includes robotic harvesters, instruments for adding samples and probes to slides, slide scanners for cell imaging, and software that can aid technologists in analysis. Looking ahead, new technologies continue to appear on the horizon, such as optical genome mapping as discussed in the July/August issue of MedicalLab Management. However, all laboratories and lab groups are different and will adopt technologies at different rates. Thus, as with all laboratory operations, reliance on skilled technologists, technicians, and other support staff will remain vital in the cytogenetics laboratory.

Virginia C. Thurston, PhD, DABMG, is the director of the Parke Cytogenetics Laboratory at the Carolinas HealthCare System in Charlotte, North Carolina. The Parke Cytogenetics Laboratory performs chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray testing for over 50 hospitals. After earning her PhD from the University of Alabama at Birmingham, Jennie completed her clinical cytogenetics fellowship at Indiana University School of Medicine (IUSM). Following her fellowship, she joined the faculty and became the assistant director of the IUSM cytogenetics laboratory. While at IUSM, Jennie also was vice chair of education for the department of medical and molecular genetics, wherein she served as course director of medical genetics for the medical school and clinical cytogenetics for the graduate school. After leaving IUSM, Jennie established and directed the BayCare Cytogenetics Laboratory in Tampa, Florida.


  1. Arsham MS, Barch MJ, Lawce HJ, eds. The AGT Cytogenetics Laboratory Manual. 4th ed. John Wiley and Sons; 2017.
  2. American College of Medical Genetics and Genomics (ACMG). Technical Standards for Clinical Genetics Laboratories. 2021 Revision; Section E. Accessed 8/31/2021: Technical Lab Standards-Section E.pdf
  3. The College of American Pathologists (CAP). CAP Accreditation Program: Cytogenetics Checklist. 2017.
  4. Wiktor AE, Van Dyke DL, Stupca PJ, et al. Preclinical validation of fluorescence in situ hybridization assays for clinical practice. Genet Med. 2006;8(1):16 –23.
  5. Wolff DJ, Bagg A, Cooley LD, et al. Guidance for Fluorescence in Situ Hybridization Testing in Hematologic Disorders. J Mol Diag 2007;9(2):134-143. doi:10.2353/jmoldx.2007.060128
  6. Mascarello JT, Hirsch B, Kearney HM, et al. Section E9 of the American College of Medical Genetics technical standards and guidelines: fluorescence in situ hybridization. Genet Med; 2011;13(7):667-75.
  7. McGowan-Jordan J, Hastings RJ, Moore S, eds. ISCN 2020: An International System for Human Cytogenomic Nomenclature. Karger International; 2020.
  8. American Medical Association. CPT 2020 Professional Edition. American Medical Association; 2019.


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