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6 Keys To Running A Proper Clinical Flow Cytometry Experiment

Written by Tim Bushnell, PhD

Clinical use of flow cytometry has paralleled the development of instrumentation and reagents.

One early application for flow cytometry is the measurement of DNA content. 

Malignancies and neoplasms often have abnormal amounts of DNA, and this can be assessed with a variety of protocols and dyes.

Comparing DNA Index (DI) of a known 2N control to a sample can yield useful information, but the clinical application of this information was limited, as it was not known how it turned these data into meaningful biological and clinical data insights.

The Rise Of Clinical Flow Cytometry

With the increased development of fluorescently conjugated monoclonal antibodies came more applications with potential clinical impact. For example, in bone marrow transplantation, studies using hematopoietic cytokines made it feasible to gather stem cells from peripheral blood.

It was also shown that reconstitution of bone marrow was accelerated when using cells from peripheral blood rather than bone marrow.

To arrive at a dose for each patient, physicians were using a total nucleated cell count per kilogram. 

But since the number of stem cells in peripheral blood is highly variable from donor to donor, and the first efforts at enumeration by culture took 10-14 days.

In the late 80’s it was shown that the majority of the colony forming and engrafting cells were contained in a small population of cells expressing the marker CD34. Now we have an assay and gating strategy that can generate accurate CD34+ counts in about an hour, greatly increasing the accuracy and ease of planning of apheresis schedules.

Multi-Parameter Instrumentation And Immunophenotyping

The area that has made the most use of today’s multi-parameter instrumentation and variety of fluorescent reagents is the immunophenotyping of hematopathologies, especially leukemias and lymphomas. 

Twenty years ago, acute lymphoblastic leukemias were typed simply as B, T, or null.

Now, B and T cell leukemias are further characterized to indicate maturation state, and these patterns of surface markers can also show correlation to specific genetic abnormalities.

In the world of hematologic malignancy, flow cytometry has made quite an impact over the decades. Under the pathologists microscope B cell Acute Lymphoblastic Leukemia (B-ALL) without maturation can look very much like B-ALL with maturation.

But in flow cytometers, we can much more quickly show that this is B cell disease rather than T cell or Myeloid malignancy.

Even better, we can define maturation state that may affect prognosis and treatment.

In the case shown below, taken from a report by Tsao, et al., you can see that even based on side scatter and CD45 something is amiss.

Where there should be 3 blood populations, Lymphs, Monos, and Grans, we see 4. 

These CD45 dim cells are leukemia blasts.

Immunophenotyping by Flow Cytometry | Expert Cytometry | Lymphs, Monos, and Grans

Looking more closely at these blasts as shown in the other plots, we can see that they are brightly positive for CD19, surface immunoglobulin, and CD20. CD20, as it turns out is a very important marker prognostically.

This is one indicator of mature B-ALL, making prognosis poor when following the traditional chemotherapeutic cycles used for B-ALL.

However, more intensive chemotherapy given along with Rituximab (as seen here) can improve outcome. Regardless of whether you misdiagnose mature B-ALL positively or negatively, when someone’s health is on the line you do not want to make mistakes.

6 Keys To Running Clinical Flow Experiments

1. The most important skill is to become VERY familiar with the patterns of normal samples so that you recognize abnormal samples. Run plenty of normal controls.

2. Keep your cytometer in excellent working order. Besides running normal human samples, run your alignment check beads, and perhaps the commercially available QC cell samples from Streck.

3. Know what markers are important for your lab. The Bethesda Consensus has a list of consensus reagents that are agreed to be important.

4. From that consensus, your lab could use a shotgun approach, using many antibodies in many parallel tubes to exhaustively characterize each sample. While costly, this means rarely needing to restain a sample.

5. Or, you might choose to use a screening approach in your lab, looking to use fewer antibodies at first to narrow down malignancy, and then retaining for further differentiation.

6. Make sure your data is de-identified and secure. HIPAA is serious business. Password protect, and if possible, encrypt your computers.

To learn more about performing proper clinical flow cytometry experiments and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Tim Bushnell, PhD

BOOKS

Advanced Microscopy

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.
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Modern Flow Cytometry

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more. 

Advanced 4-10 Color Compensation

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.

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