Diagnosis of acute lymphoblastic leukemia

The diagnosis of acute lymphoblastic leukemia is made based on the patient's history, physical examination, and laboratory tests.

Laboratory diagnostics

Complete blood count: white blood cell count may be normal, decreased, or increased; blast cells are often, although not always, detected; hyporegenerative normochromic anemia and thrombocytopenia are characteristic.

Biochemical blood test: characteristically increased LDH activity; indicators of kidney and liver function are also determined.

Myelogram: bone marrow puncture should be performed from at least two points (in children under 2 years of age, these are the heel bones or tibial tuberosities, in older children, the posterior and anterior iliac spines) to collect a sufficient amount of diagnostic material. It is advisable to collect the material under general anesthesia. It is necessary to make 8-10 smears from each point, and also collect material for immunophenotyping, cytogenetic and molecular genetic studies.

A spinal puncture is a mandatory diagnostic procedure performed by a specialist under sedation and with the presence of at least 30,000 platelets per µl in the peripheral blood (if necessary, platelet mass transfusions are performed before the puncture). At least 2 ml of cerebrospinal fluid is required to prepare a cytopreparation.

Instrumental diagnostics

It is advisable (and if there are neurological symptoms, mandatory) to perform a CT scan of the brain.

Ultrasound examination allows to determine the size of infiltrated parenchymal organs and enlarged lymph nodes of the abdominal cavity, pelvis and retroperitoneal space, the size and structure of the testicles.

Chest X-ray reveals mediastinal enlargement and pleural effusion. Bone and joint X-ray is performed as indicated.

To clarify the diagnosis and exclude heart damage, electrocardiography and echocardiography are performed. Consultations with an ophthalmologist and otolaryngologist (examination of the fundus, paranasal sinuses) are recommended.

Special diagnostic methods

Diagnosis of acute lymphoblastic leukemia is based on the assessment of the tumor substrate - bone marrow, cerebrospinal fluid.

Cytological examination of bone marrow reveals hypercellularity, narrowing of normal hematopoietic sprouts and infiltration by tumor cells - from 25% to total replacement of bone marrow by tumor.

Morphological similarity of malignant lymphoblasts and normal progenitor cells requires determination of the percentage of lymphoblasts in Romanovsky-Giemsa-stained bone marrow smears. Morphological classification of acute lymphoblastic leukemia, according to the criteria of the FAB group (French-American-British Cooperative Group), provides for subdivision of blasts into groups L1, L2 and L3 based on determination of size, structure of the nucleus, presence of inclusions and other features. More than 90% of cases of acute lymphoblastic leukemia in children are classified as L1, 5-15% as L2, less than 1% as L3. Currently, acute leukemia with mature B-phenotype (L3) is classified as a group of non-Hodgkin's lymphomas (this variant is not considered in this section).

Cytochemical examination is the next mandatory stage of diagnostics. Cytochemical staining reveals the cells' belonging to a certain line of differentiation. Myeloperoxidase staining is mandatory (the reaction of cells belonging to the lymphoid line of differentiation is negative). The PAS reaction to glycogen helps differentiate lymphoid blasts due to the characteristic granular staining of the cytoplasm. Sudan black staining is positive in myeloid cells with a typical arrangement of granules. Acid phosphatase is detected in T-cell leukemia.

Immunophenotyping is one of the main studies that determines the cellular affiliation of the blast population and the prognosis of the disease. Specific surface and cytoplasmic antigens of T and B lymphocytes are used as markers for identification, determination of the origin and differentiation stage of lymphoid cells. The use of a panel of monoclonal antibodies to differentiation clusters and determination of the percentage of their expression in the dominant population allows us to indicate whether the leukemic clone in a given patient belongs to the T or B line. According to the modern classification, the diagnosis of acute lymphoblastic leukemia is based on the results of immunophenotyping of the dominant cells.

Cytogenetic and molecular genetic methods have been widely used in recent years to study leukemic cells. The methods allow us to assess the state of the chromosomal apparatus - the number of chromosomes and their structural changes (translocations, inversions, deletions). Cytogenetic abnormalities and DNA index (the ratio of the amount of DNA in leukemic cells and in cells with a normal diploid karyotype) are significant prognostic factors. Detection of clonal abnormalities characteristic of a given patient's tumor cells allows us to track the number of these cells in the dynamics of the disease at the molecular genetic level and determine the minimum residual cell population. Identification and molecular characterization of genes whose regulation or function may be damaged as a result of chromosomal changes contributes to an understanding of the molecular basis of malignant transformation.

An important prognostic factor is the assessment of minimal residual disease, i.e. the assessment of the number of residual leukemic cells in a patient in remission. The technique for detecting minimal residual disease involves identifying cells with karyotype abnormalities using cytogenetic methods (one abnormal cell per 100 normal cells can be detected) or polymerase chain reaction (PCR allows one abnormal cell to be detected per 10 ⁵ normal cells). A very sensitive method is flow cytometry, which allows detecting cells with an abnormal immunophenotype. A high level of minimal residual disease after induction of remission or before maintenance therapy correlates with a poor prognosis.

Prognostic factors for the outcome of therapy in acute lymphoblastic leukemia

Factors	Favorable prognosis	Poor prognosis
Age	Over 1 year and under 9 years	Under 1 year and over 9 years
Floor	Female	Male
Leukocytosis	<50,000 in µl	>50,000vmkl
DNA index	>1.16	<1.16
Number of chromosomes in power cells	>50	<45 (especially 24-38)
Response on the 8th day of treatment	No blasts in the blood	There are blasts in the blood
CNS status	CNS1	CNS 2 or CNS 3
Cytogenetics	Trisomy (+4) or (+10)	T(4;11), t(9;22)
Molecular genetics	TEL/AML1	MLL rearrangement
Immunophenotype	B-predecessors	T-cell

• CNS - central nervous system.
• DNA - deoxyribonucleic acid.
• CNS 1 - absence of blast cells in the cerebrospinal fluid.
• CNS 2 - blast cells in the cerebrospinal fluid in the absence of cytosis (<5 cells per µl).
• CNS 3 - blast cells and cytosis in the cerebrospinal fluid (£5 cells per µl).

Neuroleukemia

Leukemic cells can enter the CNS from the systemic circulation, by migration through the venous endothelium and from petechial hemorrhages (profound thrombocytopenia at the time of diagnosis of the disease is associated with a high frequency of neuroleukemia). According to an alternative hypothesis, leukemic cells can spread directly from the bone marrow of the skull bones to the subdural space and then to the CNS through the adventitia of venules and nerve sheaths. Knowledge of the specific mechanism of cell penetration may have clinical applications: in cases of direct penetration of cells from the bone marrow into the CNS, local treatment is most effective, not only cranial irradiation, but also intrathecal administration of chemotherapy. In the case of dissemination of leukemic cells from the systemic circulation, systemic polychemotherapy is of greater importance. The mechanism of tumor cell penetration into the CNS depends on the type of leukemic cells, their number in the systemic bloodstream and the presence of hemorrhagic syndrome, the patient's age and the maturity of the blood-brain barrier. It is in the CNS that the overwhelming majority of tumor cells are outside the mitotic cycle; these cells can persist in the cerebrospinal fluid for a very long time - for decades. The presence of just one blast cell in 1 μl of cerebrospinal fluid means that the number of these cells in the entire cerebrospinal fluid space is at least 10 ⁵

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