Currently, according to the World Health Organization classification criteria for leukemia, cytomorphological (M), immunological (I), genetic (C) and molecular biological (M) analysis methods are needed to make a more accurate diagnosis and classification, to better help further select treatment options, to determine the intensity and duration of treatment, to judge the efficacy, and to estimate the future outcome (prognosis) of treatment.
Morphologic analysis is the observation of bone marrow and blood cells, or bone marrow tissue morphology and structure by a technician under a microscope. Morphological methods are easy, fast, and economical, and are the most basic diagnostic method for malignant blood diseases and are commonly used in hospitals. In most cases, doctors mainly take a small amount of bone marrow cell suspension to observe the cell morphology and then perform cytochemical staining. Sometimes, in order to understand the structure of the various tissues of the bone marrow tissue, the proportion of hematopoietic tissue, and to observe some cells that are difficult to extract, the doctor takes small pieces of bone marrow tissue (called biopsies) for pathological examination. Morphological methods can diagnose more than 90% of acute leukemias, and some preliminary estimates can be made for chronic leukemias. The diagnosis of acute leukemia is determined primarily by the proportion of primitive cells to all nucleated cells, so some early acute leukemias can be missed if the diagnosis of acute leukemia is based solely on this criterion. Morphologic methods are less accurate for typing acute leukemia. It has been reported that seven top hematology morphologists from France, the United Kingdom, and the United States (FAB) typed the same acute leukemia specimens and only agreed on the typing of about 70% of the specimens, meaning that about 30% of the cases were difficult to correctly typed by morphologic analysis. The error is even greater if the technician is a general morphologic analyst. Moreover, it is difficult to accurately diagnose and type chronic leukemia using morphological analysis alone.
Immunological analysis method mainly uses multiple monoclonal antibodies to label the antigens of the cells, then flow cytometry to detect multiple markers on the cell surface or inside the cells and store the data into the computer, and finally analyze these data with some analysis software to classify the cells into groups. By using immunological analysis on top of cell morphological analysis, the accuracy of typing for acute leukemia can reach more than 90%. It is possible to distinguish accurately whether the malignant cells are derived from T and B lymphocytes, myeloid cells, and it is possible to differentiate acute leukemia from chronic leukemia. Immunological analysis plays a key role in the diagnosis of some chronic lymphocytic series diseases and their staging. Morphologically, it is difficult to distinguish chronic lymphocytic leukemia and some low-grade malignant lymphomas from benign lymphocytosis such as viral infections, whereas immunological analysis can better differentiate whether the cells are benign or malignant.
Immunoassay can also detect a very small number of malignant cells (also known as microscopic residual lesions), and if there is one malignant cell out of 10,000, it may be detected by flow cytometry. Since most leukemia cells have some abnormal immunological markers, this method can be used to detect residual leukemia cells in most patients.
Genetic methods are used to find out whether malignant cells have chromosomal abnormalities by analysis of the karyotype of myeloid leukemia cells or by detecting certain chromosomal sites with probes. The chromosomal analysis of patients is increasingly valued by clinicians and researchers for its independent diagnostic value, its value in helping to select treatment, and its value in predicting the efficacy of treatment. For example, to diagnose chronic myeloid leukemia one must have t(9;22) or abnormalities involving chromosome 22, or have these abnormalities forming a bcr/abl fusion gene, otherwise the diagnosis cannot be made. If a patient has some chromosomal abnormalities specific for acute leukemia, such as t(8;21), t(15;17), and inv(16), an early diagnosis of acute leukemia can be made even if the primitive cells do not meet the criteria for acute leukemia. Then, for example, acute promyelocytic leukemia with t(15;17) chromosomal abnormalities is treated well with vincristine and arsenic, and patients with t(8;21), inv(16) chromosomal abnormalities do well with high-dose cytarabine and can be treated without first choosing transplantation; while acute lymphoblastic leukemia with t(9;22) chromosomal abnormalities, patients with complex chromosomal abnormalities, -5, and -7 and other chromosomal abnormalities are very ineffective with chemotherapy and should be treated with hematopoietic stem cell transplantation as soon as possible. Therefore the latest WHO typing protocols for malignant hematological diseases put chromosomal and/or genetic abnormalities in a very important position. Genetic analysis methods are the most specific tests, and once a clonally abnormal chromosome is detected, there is a high degree of certainty that the cell has an abnormality. However, many malignant cells cannot be detected to have abnormal chromosomes.
Molecular biology analysis can detect the presence or absence of genetic abnormalities in leukemia cells. As with genetic methods, molecular biology typing has independent diagnostic and prognostic value. Some chromosomal abnormalities can give rise to genes specific to leukemic cells, so it is possible to respond to chromosomal abnormalities by detecting these leukemia-specific genes, such as t(9;22) translocations that give rise to bcr/abl fusion genes, t(15;17) translocations that give rise to PML/RARĪ± fusion genes, and t(8;21) abnormalities that give rise to AML1/ETO fusion genes. Moreover, sometimes leukemia cells are chromosomally normal and molecular biological analysis can detect some abnormal or mutated genes, such as FLT3, WT1, NPM, etc. These genetic abnormalities can help to select the treatment and judge the treatment effect and future development. Molecular biology analysis is the most sensitive method, detecting one abnormal cell in every 100,000 cells, but the specificity is not as good as genetic analysis. Moreover, each abnormal gene must be studied and analyzed for gene sequence before a clinical routine test can be designed.
In conclusion, each of the above methods has its own advantages and disadvantages, and each method can reflect the characteristics of cells from different aspects, which can be integrated to reflect the characteristics of cells more comprehensively and better help to select treatment, judge the prognosis and judge the efficacy of treatment.