Our flow cytometry laboratory performs diagnostic and monitoring analyses as well as translational research and academic collaborations. On the diagnostic and monitoring side, we analyze peripheral blood and bone marrow, solid tumor, and spinal fluid samples. We assess children with suspected hematologic malignancies for leukemia-associated phenotype (LAP) markers. We can then monitor their progress through treatment, track their minimal residual disease, and conduct follow-up testing for potential relapse. We also monitor chimeric antigen receptor T cell (CAR-T) therapy patients for treatment response and potential relapse of disease. CAR-T cells target a specific epitope, typically CD19 or CD22 in B cell leukemias or alternative epitopes in solid tumors. But following a period of successful response to therapy, there is always the potential for relapse – and the relapsed disease can then evolve to stop expressing its target epitope. This loss of expression affects the way we have to analyze the resulting disease; gating strategies have to change, which involves using different, potentially non-lineage-specific markers. We can do this down to two cells in a million where phenotypic aberrances are pronounced. Detecting returning disease at such low levels allows for changes in disease management and therapy with greater effect than waiting for relapse to become clinically frank.
In addition, we run biomarker tests for diseases such as neuroblastoma. It’s a cancer, prevalent in pediatrics but nonexistent in adults – and it has a very poor prognosis. We want to work out how to detect it at low level, or when it has infiltrated into the bone marrow (as this alters the disease staging and treatment). We also monitor the efficacy of CAR-T therapies that might improve outcomes for neuroblastoma patients.
Bone marrow characterization
There is little published data on normal pediatric bone marrow maturation across multiple lineage and maturation markers. It has mostly been characterized in murine models, so we have needed to establish our own pediatric libraries to map hematopoiesis in different pediatric age groups. To characterize bone marrow, we look at the maturation of myeloid, monocytic, NK, T, and B cells – from the youngest cells to full maturation. This knowledge allows for greater confidence in detecting very low-level aberrant populations that do not fit typical maturation patterns.
When we’re hunting for minimal residual disease or low-level malignancy, we’re looking for something that doesn’t quite express as it should – aberrantly, or asynchronously. Our flow cytometer essentially comes with “empty” software – and we build each experiment in accordance with our needs. Basically, we use an antibody that attaches to an antigen site on a cell specific to a lineage or a maturation point. The antibody has an attached fluorochrome that excites and emits energy at a particular spectrum. When the cell goes through the flow cytometer, different lasers excite the various fluorochromes according to their individual emission spectra. We can currently look at up to 18 different antibodies with attached fluorochromes on a single cell type. Once we’ve completed our analysis, we map the results into lineage and maturation hierarchies. And that’s how we build the software; we tell it what we want to look at, and then we start mapping the outcomes of our experiments.
Now that we’ve established each lineage and its maturation patterns, we can characterize blood and bone marrow samples. Which markers are expressed? Are they all expressing normally? We look for even the smallest anomaly – two cells in a million that aren’t right – because it helps us diagnose patients, stratify them according to their risk of treatment resistance or relapse, and monitor them during and after treatment. I know that our work makes a huge difference to the patients; it allows them to receive the customized treatment they need for their specific disease. And that’s very important to us.
In Service to Our Smallest Patients
Enzymology: A Whistle-Stop Tour of GOSH Pathology
Microbiology: A Whistle-Stop Tour of GOSH Pathology
Histopathology: A Whistle-Stop Tour of GOSH Pathology
Flow Cytometry: A Whistle-Stop Tour of GOSH Pathology
Rapid response: A Whistle-Stop Tour of GOSH Pathology
Sarah Inglott is Flow Cytometry Lead Healthcare Scientist at Great Ormond Street Hospital.
