Flow cytometry for the study of hematologic malignancies

By: Dr. Vanessa Santiago Pacheco

Medical Pathologist, Laboratorio Clínico Hematológico S.A. Medellín, Colombia.

Published on 23-07-2021

Visualization of cell populations in marrow blood, according to their cytoplasmic complexity (SSC-A) and CD45 (Leukocyte Common Antigen) expression.

Flow cytometry is a rapid, reliable and inexpensive tool to establish the monotypic or polytypic origin of a cell population, which allows its use to assess marrow involvement by lymphomas and other hematopoietic diseases ^7.

Immunophenotyping by flow cytometry is an essential component in the diagnosis of acute leukemias; it allows rapid and comprehensive assessment of relevant membrane and intracellular antigens expressed by leukemic cells according to their maturation stage and progeny, which facilitates the identification and characterization of blasts, cell lineage assignment, assessment of potential therapeutic targets and prediction of some genetic alterations ^8.

Peripheral and medullary blood are the samples most commonly analyzed by flow cytometry; however, any tissue in which individual cells can be isolated in suspension can be analyzed in this way, e.g., cerebrospinal, pleural and ascitic fluid ⁹, even lysed samples of solid tissues such as lymph nodes.

Applications

Flow cytometry is a laboratory test, with rapid processing, high analytical sensitivity and specificity, that allows the characterization of cell populations in a sample; with applications in the accurate and timely diagnosis, classification, prognosis and treatment monitoring of numerous hematological malignancies.

This technology simultaneously measures and analyzes multiple characteristics of cells as they are suspended in a system fluid and pass through a beam of light, such as relative size, relative complexity or granularity, and relative fluorescence intensity. These characteristics are determined using an optical-electronic system that records how the particle (or cell) scatters light or emits ^{fluorescence1}.

This method of study has been employed for more than 40 years in the diagnosis and monitoring of hematopoietic neoplasms ^2,3, through the use of fluorochrome-labeled monoclonal antibodies, which are each directed against a specific molecule on the surface, cytoplasm or nucleus of leukocytes and hematopoietic precursors ^4.

Initial studies were performed with single antibodies or a combination of two antibodies, which limited the sensitivity for detecting minority cell populations. With the availability of new fluorochromes and stains, current analysis is routinely done with 8 to 12 different antibodies in a single sample tube4^,5,6.

Advantages and limitations

Flow cytometry has proven to be a very useful and versatile tool in the diagnosis and monitoring of hematological diseases, in addition to numerous other applications. Advances in electronics, software and reagents in recent years have simplified some aspects of flow cytometry and made it possible to combine different antibodies in a single sample tube, which in turn has facilitated the identification of abnormal populations ^9. This is a very sensitive and objective technique that allows the analysis of a large number of cells in a short time (5,000 to 20,000 events/second) and the use of more than one antibody at a time to identify subpopulations within a heterogeneous population. This ability to acquire large numbers of cells (events) has made it possible to detect low numbers of abnormal cells, which may persist after treatment, to measure minimal residual disease and to add these data to personalized treatments ^8. Historically, flow cytometry has been perceived as a highly experience-dependent technique with low reproducibility; however, consensus building has allowed the development of standardized panels for rapid and accurate diagnostic approach, in addition to the classification of hematological malignancies and their follow-up, such as the Euroflow® consortium ^10. Another limitation that has been described in the study of flow cytometry is the absence of a morphological assessment of neoplastic cells, since this is an essential part of the diagnosis and classification of hematopoietic diseases. This is why immunophenotype assessment should be performed in conjunction with observation and analysis of the biopsy of the affected tissue, such as peripheral blood smear, bone marrow biopsy and aspirate, or lymph node biopsy. In the ideal context for the patient, these samples should be evaluated by the same specialist to arrive at a comprehensive diagnosis.

The Laboratorio Clínico Hematológico in the classification of hematological malignancies.

We have flow cytometry for bone marrow, peripheral blood, fluid and tissue biopsy samples, adjusted to the protocols and panels determined by the Euroflow® ^4. consortium and with access to its databases to achieve a reproducible diagnosis. Our turnaround time for results is two (2) working days, with immediate telephone reporting in case of obtaining critical values, such as the diagnosis of acute leukemias or high grade lymphomas. From the Diagnostic Hematology Division, we offer an Integrated Hematopathology Report, both for our own cases and for the specialized interconsultation service; a report that, beyond the report of histopathological and immunophenotypic findings, provides a complete and specialized analysis of each patient in a global context. This, in order to provide a clinical, morphological and laboratory correlation that allows the treating physician a practical approach that adds value to the management and clinical benefit of the patient. We guarantee direct contact and teamwork with our specialists in hematopathology, for the discussion and joint analysis of cases of difficult approach or management, and for the immediate reporting of urgent cases or acute disease. In addition, we offer the possibility of remote interconsultations for complex cases with international experts in the area of hematopathology, laboratory hematology and flow cytometry.