leukemia diagnosis What is Leukemia?

 
Leukemia: Diagnosis
What is Leukemia?

Leukemia is a neoplasm of the blood characterized by the proliferation and accumulation of tumor clones in the bone marrow, peripheral blood and lymphoid organs. Diagnosis of Leukemia The disease, suspected on the basis of symptoms and physical examination, is confirmed through laboratory investigations and instrumental examinations. In particular, the analysis of peripheral blood (blood count) and bone marrow (taken through a needle aspiration) allows to identify the tumor cells and define their characteristics. Other tests to confirm the diagnosis of leukemia are radiological investigations to evaluate the enlargement of the liver and spleen, and the possible involvement of other organs.
Objective examination

The diagnosis is always preceded by the detection of the patient's clinical data (anamnesis) and a physical examination, through which the possible presence of enlarged lymph nodes or the increase in volume of the liver and spleen is sought. In addition, the medical examination allows to evaluate: general conditions, fever, sweating, weight loss, infections, previous anemias or hemorrhagic episodes.
Blood test

Complete blood count and morphological evaluation by peripheral blood smear are fundamental for diagnostic orientation.

    Complete blood count
        Cell count: number of red blood cells, leukocytes and platelets.
        Hb level.
    Peripheral blood smear
        The peripheral blood sample, taken from the patient and sent to the analysis laboratory, is submitted to a morphological examination under a microscope to ascertain the presence of blasts.
    Determination of haematochemical parameters: azotemia, glycemia, transaminases, etc.
    Biochemical profile for renal function, liver enzymes and bilirubinemia, uricemia, LDH, beta-2-microglobulinemia (indicators of functioning of kidneys and liver).

In the case of leukemia, through the blood test, in general, we highlight:

    Anemia: decrease in the concentration of hemoglobin and the number of red blood cells;
    Thrombocytopenia: decrease in the number of platelets;
    Leukocytosis: increase in the number of leukocytes (less frequently, a condition of leukopenia is observed, with a decrease in the number of white blood cells).

Interpretation of the blood test
Reference note: Acute lymphoblastic leukemia = LLA; Acute myeloid leukemia = LMA; Chronic lymphatic leukemia = LLC; Chronic myeloid leukemia = CML.

    Most patients show some anomaly in the blood count. The peripheral smear allows to highlight the presence of blasts in patients with acute leukemias. In the characterization of the forms of LLA it is necessary to resort to the application of immunological techniques for a complete diagnostic definition, unlike LMA, where the morphology and the cytochemistry are sufficiently indicative, to discriminate the different subtypes.
    A lymphocytosis of variable degree (high number of lymphocytes between 10,000 and 150,000 / mm3) must be present to diagnose the CLL. The absolute neutrophil count is usually normal; the number of red blood cells and platelets has slightly decreased. According to the criteria codified by the FAB group (French-American-British, which organizes morphological and cytochemical characters in schemes that allow to classify different types of leukemia), a condition to confirm the diagnosis of LLC is represented by the presence of atypical lymphocytic elements (prolyphocytes) , immunoblasts and lymphoblasts) less than 10% in the leukocyte formula. Moreover, at the peripheral striation it is possible to detect mature lymphocytes with poor and non-granular cytoplasm, and the presence of the Grumprecht shadows (expression of rupture of the trauma cells, typical of the LLC).
    The CML is defined with the white blood cell count: the haemochromocytometric examination shows a leukocytosis that can vary from 20 to 300 x 109 / l WBC (WBC = number of white blood cells per liter of blood). Morphological evaluation of peripheral blood reveals mature and immature elements of the neutrophilic granulocyte series and an increase in the number of eosinophils, monocytes and / or in particular basophils is often observed. Unlike the leukemic clones of LMA, these cells are mature and functional. The number of platelets may be normal (in 60% of cases), increased (30%) or reduced. A picture of modest anemia may be accompanied by findings of leukocytosis and / or thrombocytosis. The leukocyte alkaline phosphatase is generally reduced or absent. Other laboratory findings useful for diagnosis may be represented by the generally high levels of uricemia and LDH in serum.
    To classify the LMA, use of appropriate panoptic stains (allow simultaneous observation of all blood cells) of peripheral blood smears


Bone marrow and rachicentesis examination

The bone marrow can be taken in two different ways:

    Osteomedollary biopsy
    Medullary needle aspiration

Both procedures, performed under local anesthesia, consist of a bone puncture (at the level of the iliac crest, the sternum or the femur) to take a small amount of blood from the bone marrow, and a small bone fragment in the case of biopsy .

The doctor, using the microscope, will examine the sample to try to identify the presence of tumor cells: the medullary needle aspiration allows to perform a cytological examination, while the biopsy allows to perform a histological characterization. The collected bone marrow sample can also be subjected to other diagnostic investigations: morphological examination (microscopic identification of the blasts), cytochemistry, flow cytometry, cytogenetics and molecular biology. The aspirated bone marrow and the bone marrow biopsy allow to identify the type of leukemia and to define the type of therapeutic strategy to be adopted.

A diagnostic investigation that is sometimes used to deepen the evaluation of acute lymphoblastic leukemia and acute myeloid leukemia, is the rachicentesi, which consists of a lumbar puncture (in the lower back); by means of a fine needle inserted between the last two vertebrae, a sample of cerebrospinal fluid is taken (a liquid that fills the spaces around the brain and spinal cord). The liquor sample will be examined in the laboratory, looking for tumor cells or other signs of alteration.
Interpretative notes on the examination of the bone marrow

    Analysis of a bone marrow sample establishes the diagnosis of leukemia. The morphology of the blasts makes it possible to distinguish between LLA and LMA.
        The bone marrow, in the LLA, is generally presented with a homogeneous and conspicuous infiltrate by the lymphoblasts, small and with poor cytoplasm, which replace the normal elements of the bone marrow. For the diagnosis of AML, 30% of the nucleated cells in the aspirate must be blasts of myeloid origin.
        Myeloblasts are characterized by the bodies of Auer, which are multiple groupings of blue-gray granular material, forming elongated needles, visible in the cytoplasm of the leukemic clones. The presence of Auer's bodies is diagnostic for the LMA, since these structures do not appear in the LLA.
    In the LLC, the medullary needle aspiration shows a lymphocyte infiltration variable between 40% and 95% of the total cells.
    In case of CML, the medullary aspirate reveals a marked hypercellularity with hyperplasia of the granulocyte and often also megakaryocyte series. Bone marrow biopsy confirms myeloid hyperplasia with marked reduction of the erythroid compartment and almost complete disappearance of the adipose component. The weft of the reticular fibers of the bone marrow may be normal or slightly increased (the medullary fibrosis correlates to the more advanced stages of the neoplasm).

Immuno-phenotypic analysis

The multiparametric flow cytometry, applied to the cells present in a blood or bone marrow sample, allows to characterize in a deeper way the cell population involved in the pathology: the immunophenotyping, following labeling with monoclonal antibodies, allows to identify specific antigens of surface, allowing therefore the typification of the clones (distinguishes, for example, the monoclonal expansion B or CD5 + in the LLC).
Interpretative notes on immunophenotypic analysis

    In lymphoid leukemias, the determination of the immunophenotype allows the characterization of the lymphocytes: with the cytofluorimetry the origin of the lymphoblasts is identified (distinguishes the B cells from the T). LLC expresses some surface antigens such as CD38, CD19, CD20, CD23, CD52 etc. Moreover, the cytometry allows the demonstration of the presence of surface Ig and of the monoclonal expression in the lymphoid leukemias (example: all the cells express only light chains of Ig of type κ or only of type λ). The tumor cells correspond to a minor subpopulation of B cells that express on the cell surface immunoglobulin M (IgM) and immunoglobulin D (IgD) or the CD5 + antigen, associated with T clones.

Some specific antigens of the myeloid lineage, such as CD13, CD33, CD41 etc. they have been used to diagnose LMA: the determination of the immunophenotype through the use of monoclonal antibodies shows more or less specific surface and / or cytoplasmic markers, which allows to identify the different stages of cell differentiation.

Cytogenetic and molecular analysis

In the laboratory we examine the chromosomes, genes and the expression of the transcripts, obtained from blood cells, bone marrow or lymph nodes, to establish the type of leukemia.

    Conventional cytogenetic analysis (karyotype reconstruction): investigation that detects the presence of chromosomal abnormalities in pathological cells. This analysis recognizes the "primary" anomalies (present in all the abnormal cells), responsible for the early stages of transformation. Identifies the "secondary" alterations responsible for the phases of clonal evolution. It must identify lesions not relevant to the pathogenesis of the disease, as it is a simple expression of genetic instability.
    Molecular cytogenetic analysis: FISH (fluorescent in situ hybridization) is a research that combines the competence of cytogenetics and molecular techniques. The fluorochrome-labeled probes allow to detect in the chromosomes or in the interphase nuclei the presence of a DNA sequence of the order of magnitude between tens and hundreds of Kb.
    Molecular biology techniques: PCR (sensitive analytical technique, which detects the presence of "rare" cells), RT-PCR (PCR preceded by inverse transcription) etc.

Interpretative notes on cytogenetic and molecular analysis

    For the diagnosis of Chronic Myeloid Leukemia, cytogenetic tests are indispensable. The Philadelphia chromosome can be seen in 90-95% of CML cases. The use of FISH (fluorescent in situ hybridization) using specific probes for BCR and ABL genes, allows to quantify the positive Ph clone. The analysis in RT-PCR defines the type of transcript BCR / ABL. In particular, the detailed analysis of the three different transcripts (p210, p190, p230), and then of the different abnormal proteins, allowed to document that these are more frequently associated with different disease phenotypes: p210 - frequent in CML, rare in LLA ; p190 - frequent in the LLA, rare in the CML, rare in the LMA; p230 - LMC with a strong presence of a mature granulocyte population.
    The LMA is characterized by numerous chromosomal abnormalities that have been and continue to be identified: these allow, in particular, to distinguish de novo (primitive onset) leukaemias from the secondary ones. The cytogenetic and molecular alterations therefore represent a precise reference to identify specific markers of the different types of LMA, important for diagnosis and for the prognostic implications.
    Cytogenetic analysis of LLA reveals the presence of clonal chromosomal aberrations in 90% of patients. 30-50% of the LLA forms present a pseudodiploid karyotype, while 30% have a hyperdiploid structure (alterations in the number of chromosomes). The structural aberrations found with more frequency are: t (9; 22), t (4; 11), t (8; 14) t (1; 19) t (11; 14) t (7; 14), 6q- .
    The cytogenetic anomalies found in the LLC include: +12 (trisomy of chromosome 12 present in 25% of cases), 14q +, structural alterations to chromosomes 13, 11, 6, 17 (in particular, deletion of the long arm of chromosomes 13, 6 and 11 and the deletion of the short arm of chromosome 17). Among the biological factors that occur are identified: the mutation of the genes that regulate the production of Ig, the expression of the protein ZAP-70 (tyrosine kinase expressed in normal T lymphocytes: one of its mutations determines a worse prognosis), the expression of 'oncogene p53.
    In LLA, the typically found abnormalities are: the translocation t (8; 21) between chromosomes 8 and 21, which determines the origin of a molecular marker called AML1 / ETO; t (15; 17) and the molecular mutation PML / RAR alfa; alterations involving the 11q23 chromosomal band and chromosome 3.

The doctor, during the diagnosis, can prescribe other analyzes, related to the manifestation of symptoms and the type of leukemia. These tests could be associated, for example, with a chest radiograph and an ultrasound of the abdomen to show a swelling of the lymph nodes or other symptoms, such as an increase in the size of the liver or spleen.