In order to differentiate between cell structures, the May-Grünwald/Giemsa stain is used (MGG stain). This staining method works by a chemical reaction between stain substances and the biochemical components of the cell. The stain substances are neutral salts which ionize in water and react with oppositely charged ions in the cell. MGG staining is the basis for the FAB classification.
Alpha-naphthyl acetatesterase staining
In many cases of MGG stained of bone marrow specimens, and even easier in peripheral blood, there may be indications of myelomonocytic leukemia (FAB classification M4-M5). In certain cases, these immature cells will have an abundance of cytoplasm, but no apparent clover leaf-cuts in nuclei, making the FAB classification ambiguous (either M1-M2 or M4-M5). In these cases, the alpha-naphthyl acetatesterase stain is useful, which is positive in myelomonocytic leukemia and monocytic leukemia and negative for other types.
Diaminobenzidine peroxidase staining
Of the cytochemical examinations, diaminobenzidine peroxidase (DAB stain) is the most useful supplement to MGG staining. Some acute myeloid leukemia, as when using the MGG stain, have under 5% promyelocytes and will have more than 5% granulated, immature/blast-resembling cells when using this stain.
Lysozymes in serum
The lysozymes are produced by cells in the granulocytic series and predominantly the monocytic series. The enzyme is not produced by cells in the lymphocytic series. If a patient with acute leukemia has a raised serum lysozyme, it is probable the patient has acute myeloid leukemia and acute lymphatic leukemia is very unlikely. If the serum lysozyme is more than 4 times over the normal limit, this indicates M4 or M5.
Flow cytometry immunophenotyping
Immunologically, the disease is defined by expression of two or more of the following myelomonocytic markers: myeloperoxidase (MPO), CD13, CD33, CD65 and/or CD117. MPO is considered to be the most specific myeloid marker. An attempt at correlating immunophenotype with subtypes with regard to the FAB classification has been made, but there are only 3 subtypes which are clearly defined by their immunophenotype: M0, M6 and M7.
Cytogenetic and molecular genetic testing
Using genetic technology methods, for example FISH analysis, (fluorescent in situ hybridization) or PCR (polymerase chain reactions), details are revealed of gene material which change places, lacks or changes of chromosomes, and the significance this has for differentiation and growth of leukemia cells. This has an increasing significance for treatment since the methods are very sensitive for the presence of characteristic gene material from leukemia cells. An example of this is testing for internal tandem duplication (FLT3 gene) which is an unfavourable prognostic sign.
FLT3 is a tyrosine kinase receptor which has a large impact on hematopoetic cell proliferation. In 18-30% of all adult patients, an internal tandem duplication (ITD) is observed of FLT3-ITD, which is considered to cause an increase of tyrosine phosphorylation leading to leukemia development.
The FLT3 mutations occur most often in acute myloid leukemia with normal karyotype and high blast value in the blood. There is agreement that FLT3-ITD is associated with high recurrence risk in younger patients. The FLT3 gene is also observed in M3,but here without indicating a poorer prognosis.
Another example is nucleophosmin1 mutation (NPM1-mutation). In patients with normal cytogenetics and absence of FL3T-ITD the presence of such mutations is associated with better than average prognosis.
Gene technology methods are increasing the understanding of leukomogenesis and thus contributing to development of new treatment principles. Testing for the APL/retinol alpha receptor, the translocation with DNA-hybridization techniques in M3, is an example of this. For this type of leukemia, very good results are achieved by treatment with retinol, which causes differentiation and apoptosis of leukemia cells.
More than half of the patients with acute leukemia have non-random (clonal) chromosome changes. That is, the same change in two or more mitoses from bone marrow cells. Certain cytogenetic changes are associated with special morphological changes.
Great emphasis is put on the results of such examinations during the risk consideration regarding transplantation of allogeneic stem cells in the first remission.