Table comparing conventional biomarkers of leukemia with MORE analysis.
(1) Morphological analysis of air-dried Romanowsky (Wright, Wright-Giemsa, or May-Grünwald- Giemsa)-stained blood or bone marrow smears. The morphological features identified by microscopic examination may suggest either lymphoid or myeloid differentiation of leukemic cells, but with the exception of the identification of Auer rods in myeloblasts none of these features is lineage-specific. Sub-clones can be identified by differences in size and morphological features (e. g. cytoplasmatic vacuoles). (2) Cytochemical staining improves the accuracy and reproducibility of lineage assessment and therefore is required for traditional sub-classification of acute myeloid leukemia (AML) according to the French-American-British (FAB) and WHO criteria. Sudan Black and stains for myeloperoxidase (MPO) to identify myeloblasts and esterase stains like alpha-naphthyl-butyrate to identify monoblasts have remained useful in this regard. Staining must be performed without undue delay as MPO is unstable and becomes undetectable after a week of storage. (3) Immunophenotypic classification is based on identification of cell surface epitopes or cytoplasmatic proteins by fluorescent dye-labeled antibodies. Flow cytometry (fluorescence-activated cell sorting, FACS) is nowadays widely used as a particularly powerful method because multiparameter analysis offers the advantage of segregating leukemic cells from non-neoplastic cells. Thus, rapid analysis allows to establish the lineage of the leukemia (e.g. myeloid versus lymphoid), its stage of differentiation (e. g. T- versus B-ALL) and facilitates minimal residual disease (MRD) monitoring using a leukemia-specific pattern of markers not expressed in that combination on regular blood or bone marrow cells. Notably, some precursor B-cell ALL might be negative for CD45 (leukocyte common antigen) or patients with T-ALL lack TdT or CD34 expression. Although ALL can be classified according to the stage of maturation, the optimal immunologic sub-classification remains a matter of debate. Many ALLs also aberrantly express myeloid-linage associated antigens (mostly CD13, CD33). Therefore the antibody screening panel for acute leukemias must be designed to include at least one very sensitive and one relatively specific marker for each hematopoietic and lymphoid lineage. (4) Molecular (genetic) classification using traditional methods will detect specific cytogenetic and/or molecular abnormalities in 60–80% of ALL and 50–60% of AML cases. The recent advent of whole genome analysis has allowed virtually all acute leukemias to be classified according to specific genetic abnormalities. Markers can be separated into leukemia-specific (e.g. BCR-ABL1; t(15;18)) or leukemic-clone specific (e.g. Ig-heavy chain gene rearrangements, T-cell receptor gene rearrangements). Both are valuable for classification, as prognostic indicators with a defined treatment applied, and are nowadays routinely used for monitoring of MRD by exploiting the high sensitivity of PCR-based amplification of specific gene sequences. The technique is time-consuming and expensive, and usually performed only in reference laboratories. (5) MORE analysis. When compared to these established conventional methods, the advantages of morpho-rheological (MORE) phenotyping are characterized by a very short time for analysis and the minimum amount of blood required. The technique has comparable power with regard to the identification of leukemic cells and the identification of leukemic sub-clones. Its applicability to classify the leukemic lineage (for example by significant differences in size, deformation, and Young’s modulus; see Figure 4—figure supplement 1) and to detect small numbers of leukemic cells can theoretically be expected and has been shown in single cases already, but still has to be tested and proven in a formal comparison, which is beyond the scope of the present study. Potentially, the rheological features of blast cells might represent additional prognostic biomarkers for leukemic cells (stiffness might correlate to drug sensitivity or refractoriness, or identify a leukemic subclone), which will be the subject of future studies. Morpho-rheological phenotyping, thus, compares very well to established biomarkers for following ALL treatment success.