Upgrading Flow Cytometry Instrumentation

May 2021 - Vol.10 No. 5 - Page #2
Category: Flow Cytometry

Having had access to flow cytometry technology since the early 2000s, it has become integral to the services offered to our patients. Now, the flow cytometry laboratory, as part of the department of pathology at The Valley Hospital in Ridgewood, New Jersey, performs three major tests: Leukemia and lymphoma immunophenotyping, T and B cell enumeration, and PNH (paroxysmal nocturnal hemoglobinuria) evaluation. These advanced tests serve to support oncology, immunology, and other hematology-related services at the hospital, but as with all leading-edge technologies, they require strict control and a close attention to clinical utility in order to optimize their impact. The abilities of modern flow cytometry technology are very enticing and a practical approach to implementing or updating this technology will reap benefits for the lab, the institution, and the patient.

Flow Cytometry Applications in Use

Although not strictly limited to these applications, the following are a sample of the most common uses of flow cytometry in our laboratories.


Leukemia and lymphoma immunophenotyping is among the more common tests used to diagnose blood-related cancers. In this case, highly specific monoclonal antibodies that have been treated with a fluorochrome are utilized to detect the presence or absence of various cellular components that are commonly seen in certain types of cancers. This information is then used in the diagnosis, prognosis, and treatment of these diseases, and to help determine whether the treatment has been successful. This capability is especially useful during early stages of malignant disease development, when a relatively scant number of cancer cells are present in the sample, and those few cells could go undetected by ordinary examination under a microscope.

Immunology & Infectious Disease

An additional common test performed by flow cytometry is the T and B cell lymphocyte subset enumeration. This test is performed to measure the strength of the immune system in a person after having been diagnosed with HIV and to monitor the effectiveness of treatment. Occasionally, it may be used with other immunodeficiency conditions, such as functional
granulocytes disorder (eg, leukocyte adhesion disorder), specific granule deficiency, and chronic granulomatous disease (CGD).

Hematological Disorders

Flow cytometry is the gold standard for testing for paroxysmal nocturnal hemoglobinuria (PNH). PNH is a rare acquired clonal hematopoietic stem cell defect with an estimated frequency of 1-10 per one million.1 The disorder results in episodic intravascular hemolysis, hemoglobinuria, hemolysis, and venous thrombosis, and is associated with aplastic anemia.

Fortunately, this technology provides a definitive diagnosis, as a recent introduction of high-sensitive flow cytometry makes it possible to detect 0.01% of PNH clone; a process we have successfully performed ourselves.

Instrumentation Upgrade

In 2019, our laboratory leadership decided to upgrade our flow cytometry instrumentation and acquisition platform, thereby introducing the possibility for increased accuracy in population identification, the ability to obtain detailed information from low cellular specimens, and the means to consistently detect abnormal populations at a low level. All of these improvements contribute to greater efficiency in the lab and faster, more reliable testing results.

In the course of this improvement, we switched from our vendor’s previous instrument that had a 1-laser, 5-color configuration, to its latest instrument, featuring 3 lasers and a 10-color configuration. The electronic design of this new instrument provides 6 levels of sensitivity and resolution to help easily distinguish a wide range of populations, including leukemia and lymphoma cells, as well as minimal residual disease (MRD).

It is worth noting that this new instrument is intended for use as the first FDA-cleared IVD 10-color panel for leukemia and lymphoma diagnostics. While relatively few clinical laboratories in the US are using this platform for diagnosis of hematological disorders, The Valley Hospital’s department of pathology and laboratory medicine is among them, and we hope this number is growing.

Correlation and Improvement

To validate the new IVD system, we correlated the immunophenotype results from our previous, 5-color leukemia/lymphoma panel with the new, 10-color IVD leukemia/lymphoma panel. The patterns of antigen expression were measured in a series of cases, such as bone marrows, peripheral bloods, lymph nodes, and fine-needle aspirations (FNAs), and included chronic leukemias, acute leukemias, non-Hodgkin lymphomas, plasma cell neoplasm, myelodysplastic syndrome, myeloproliferative neoplasm, as well as normal cases.

High correlation was observed between all parameters obtained with the 5-color and 10-color panels performed with both the original and new instrumentation. However, the 10-color system in combination with its new software allows acquisition in a shorter time and with a smaller sample amount. Furthermore, the 10-color antibody tubes eliminate the need to pipette antibodies, improving efficiency while reducing potential for human error. The use of dry, unitized reagent assays also helps meet FDA-cleared and CE-marked compliance requirements, thereby reducing the need for extensive validations.

Given the 10-color capabilities of our new flow cytometer, we can collect additional data points from each sample, consequently reducing the number of samples to prepare (and the number of possible errors that can result from manual preparation processes). Certainly, several arduous, time-consuming operator tasks often associated with the previous generation of flow cytometry instruments, such as color compensation, list mode analysis, sample repeats, and interpretations, have been substantially reduced with the new instrumentation and attendant software.


As a result of our instrumentation upgrades, we have seen a substantial benefit in flow cytometry operations. The system has helped mitigate the time-consuming, error-prone steps of lab-developed tests and also lowered overall reagent costs through increased efficiencies. By combining experienced technologists with an advanced flow cytometry IVD system designed to help detect a variety of leukemias and lymphomas, we are able to perform immunophenotype analysis on a smaller sample with better standardized compliant information and gain greater confidence and reliability in our clinical findings, all with tremendously expedited turnaround times to our health care professionals.


  1. Wagar E. Paroxysmal Nocturnal Hemoglobinuria (PNH). Labmed. Accessed 4.20.21: cancertherapyadvisor.com/home/decision-support-in-medicine/labmed/paroxysmal-nocturnal-hemoglobinuria-pnh/

Natalya Savelyev, MS, MT(ASCP) is the flow cytometry specialist at The Valley Hospital in Ridgewood, New Jersey.


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