Multiparametric flow cytometry is currently the most popular (and preferred) alternative to microscopic differential counting since it allows analyzing thousands of cells in a standardized way in a short time, with the possibility of detecting not only the populations usually recognized and classified in a blood smear, that is, neutrophils, eosinophils, basophils, lymphocytes, monocytes, immature granulocytes, and blasts, but also other subsets of cells, including various types and subpopulations of blasts and lymphocytes. In addition, recent studies have demonstrated the feasibility of flow cytometry using combinations of monoclonal antibodies that allow the detection and quantification of up to 16 cell sub sets. The instruments most commonly used in laboratories are based on two technologies, either alternative or combined, depending on the complexity of the instrumentation and the manufacturer, namely electrical impedance and cytochemistry.
The differential leukocyte count is primarily necessary for the following reasons:
• To search for quantitative anomalies in a population of morphologically normal leukocytes, such as in infectious or allergic diseases.
• To therapeutic monitoring of cytotoxic or myelotoxic drugs. Of course, this requires a high level of precision and accuracy.
• To detect morphological abnormalities of white blood cells (e.g., when abnormal populations of white blood cells are circulating or when the presence of immature or atypical cells is suspected). This requires a high level of clinical sensitivity, that is, the ability to identify all patients with abnormal circulating leukocytes.
Automated differential counting provides a high level of accuracy and precision in quantifying and identifying nor mal white blood cells. However, the method is not always sufficiently sensitive in identifying abnormal or immature cells, and it cannot identify and classify all types of white blood cells accurately. To overcome this problem, many automated analyzers flag the presence of possible abnormal white blood cell populations, thereby indicating the need for a peripheral smear examination by personnel capable of identifying abnormal cells. Monocytes and basophils are the most difficult populations to count, and as a result, counting them has a low level of precision and accuracy. In addition, automated analyzers tend to underestimate basophil counts in true basophilic conditions. It should also be noted that both automated and manual methods are unable to detect a small number of abnormal cells. The false-negative rate reported in the detection of abnormal cells varies from 1% to 20%, depending on the instrument and the required detection limit. The identification of lymphoma cells and reactive lymphocytes is the most difficult condition for both automated instruments and microscopic examination. The value of reporting the presence of banded core granulocytes is questionable. However, the measurement of immature cells in myeloid lines, particularly bands, has been considered clinically useful in the diagnosis of infections, particularly neo natal sepsis. However, it should be considered that neutrophil granulocytes with banded nuclei cannot be enumerated by automated analyzers, as they are reported together with segmented neutrophils as absolute neutrophil counts. Identification of neutrophils with banded nuclei by microscopic examination is neither precise nor consistent due to the high variability in morphological classification since quantification of banded neutrophils reflects interexaminer variability.
