Immunophenotyping of acute leukemia – goals, objectives

The introduction of monoclonal antibodies into practice allowed us to identify specific receptors, antigens and other molecules (markers) on the membrane and / or in the cytoplasm of blast cells. Currently, more than 300 antigenic groups have been identified, which are called differentiation clusters (CD).

The objectives of immunophenotyping in acute leukemia:

1) confirmation of the diagnosis established on the basis of studying the morphology and cytochemistry of the blasts;
2) establishment of a diagnosis with questionable morphological and cytochemical results;
3) diagnosis of biphenotypic acute leukemia;
4) detection of aberrant antigen expression for the detection of a neoplastic clone with signs of complete remission (minimal residual disease);
5) division of acute leukemia into prognostic groups.

Cells of acute lymphoblastic leukemia of T-cell origin are characterized by the expression of T-cell markers CD2, CD5, CD7 (less commonly CD1); sometimes B-cell markers CD10 and CD21 can also be ex-compressed. In cases of B-ALL, CD19, CD10, CD22 and, depending on the stage of maturation, CD20 and surface immunoglobulin are expressed.

In acute myeloid leukemia, CD13, CD15, CD33, and CD14 are expressed (with the monocytoid component). Expression of Tdt (terminal deoxynucleotide-diltransferase) is noted in most lymphoid blasts and in 20% of cases of acute myeloblastic leukemia. CD34 can be expressed on blast cells of all lines, especially poorly differentiated.

HLA-DR is determined on B-ALL blasts, most AML (except for MV) and in rare cases with T-ALL.

In most cases of biphenotypic leukemias (a combination of phenotypes of two and even three lines), blasts show coexpression of markers of several lines; in more rare cases, the same patient simultaneously has blasts of different origin (myeloid and lymphoid) – bilinear acute leukemia.

Classification of acute leukemia

In 1976, a group of experts from France, the USA and the UK (FAB) proposed a classification of acute leukemia, based on the morphological and cytochemical features of blast cells.

Currently, you should also use the results of immunophenotyping and cytogenetic research necessary to determine the individual prognosis and the choice of adequate therapy (intensification, myelot transplantation or standard treatment, which often includes long-term maintenance chemotherapy).

In accordance with the FAB classification, on the basis of morphological examination of blood and bone marrow and cytochemical reactions, 8 types of acute myeloblastic leukemia (AML) (M0-M7) and 3 types of acute lymphoblastic leukemia (L1-L3) are distinguished. Currently, the FAB classification of ALL is not used; in AML, immunophenotyping and cytogenetic study of blast cells are almost always used additionally.

The most important for differential diagnosis is the allocation of lymphoid or myeloid lesions of acute leukemia, since there are significant differences in the treatment of patients with AML and ALL. Due to the fact that modern therapy programs for all morphological types of AML, with the exception of ALI, are usually similar, and the prognosis is usually unfavorable, the use of only the FAB classification is impractical.

It should also be borne in mind that in some patients, morphological and cytochemical studies conducted by qualified laboratory physicians either do not allow verification of the variant of RL, or give an erroneous diagnosis in some patients (in the study of peripheral blood and bone marrow preparations by various experts, the diagnosis is confirmed no more than than in 80-90% of cases).

In these cases, immunophenotyping of blast cells is indicated. Finally, at present, cytostatic therapy programs are differentiated depending on the immunological variant (first of all in ALL) and the nature of cytogenetic changes. For all these reasons, morphological and cytochemical examination of blood and bone marrow in most cases should be complemented by immunophenotyping and cytogenetic studies.

Differential diagnosis of acute leukemia

Differential diagnosis of acute leukemia is carried out with other diseases of the hematopoietic system (non-Hodgkin’s lymphomas, myelodysplastic syndromes, blast crisis of chronic myeloid leukemia, multiple myeloma, aplastic or megaloblastic anemia), leukemoid reactions (most often associated with lymphoma cytosarcidoproteinoproteinoproteinoproteinoproteinoprostocrostostostostosidocaptophaloprooptosinopoproteinza anemia, anesthesia-anemia-anemia-anemia-anemia-anaineathrotostostostosidrotherapy syndrome, blast crisis, chronic myeloid leukemia, blast crisis). pronounced monocytosis in tuberculosis and other infectious diseases), “exit” from agranulocytosis, metastasis of solid tumors in the bone marrow.

The study of the bone marrow almost always allows to exclude non-hematological pathology, since in acute leukemia there is an increase in the content of blast cells. Detection of at least 20% of typical blast cells in a smear of peripheral blood and / or bone marrow eliminates most hematological diseases that have similar clinical and laboratory manifestations.

The exceptions are:

1) one of the variants of myelodysplastic syndrome (MDS) (refractory anemia with an excess of blasts – RAIB), which usually differs from acute leukemia only by the percentage of blast cells in the bone marrow;

2) blast crisis of chronic myeloblastic leukemia.

In 30% of patients over 60, the development of acute myeloblastic leukemia is preceded by one of the variants of myelodysplastic syndrome (MDS) (most often RIBS). In both cases, the results of treatment and the prognosis are usually unfavorable, therefore the differences between refractory anemia with an excess of blasts (RAIB) and secondary acute myeloblastic leukemia (AML) developed from myelodysplastic syndrome (MDS) do not have significant clinical significance.

For differential diagnosis with blast crisis of chronic myeloblastic leukemia (CML), it is necessary to consider the history of the disease, clinical data, the results of cytogenetic research and immunophenotyping. When formulating a diagnosis, it is necessary to isolate secondary acute leukemias (almost always acute myeloblastic leukemias (AML)) that developed after radiotherapy and / or chemotherapy for tumor and non-tumor diseases.

Myelogram with acute leukemia.

Trepanobiopsy is not a mandatory study for acute leukemia, but it is necessary to conduct it at low cellularity of the bone marrow or “dry” punctate to exclude aplastic anemia and subleukemic myelosis.

Cytological examination of the cerebrospinal fluid in acute leukemia is performed in all patients with acute leukemia before treatment. In the absence of pathology in acute lymphoblastic leukemia, myelomonoblastic and monoblastic acute leukemia, further prevention of neuroleukemia is carried out. With the development of neuroleukemia, it is treated, the results of which are evaluated on the basis of an analysis of the cellular composition of the cerebrospinal fluid.

Biochemical studies in acute leukemia. In most cases, biochemical parameters are within normal values, however, in certain cases of acute leukemia (ALL, monoblastic leukemia), impaired renal function (increased creatinine level) may be observed due to their infiltration with tumor cells. Specific renal infiltration and / or their increase can be documented using ultrasound or computed tomography. In some cases (with acute leukemia with hyperleukocytosis, acute lymphoblastic leukemia with organomegaly), tumor lysis syndrome is detected already in the debut of the disease.

More often, however, this syndrome occurs with the rapid lysis of cells during chemotherapy and is characterized by hypocalcemia, hyperkalemia, increased LDH levels, and hyperuricemia with the development of renal failure.

Instrumental studies for acute leukemia are not critical for acute leukemia, but their results can influence the nature of the treatment and the prognosis of the disease. Thus, radiography of the chest organs reveals an increase in mediastinal lymph nodes, pneumonia; electrocardiography – rhythm and / or conduction disturbances due to specific myocardial infiltration, anthracycline cardiomyopathy, etc.

Laboratory diagnosis of acute leukemia – analyzes

A blood test for acute leukemia. Most patients with acute leukemia (OL) at the time of diagnosis of the disease have normochromic normocytic anemia, which is more pronounced in acute myeloblastic leukemia. With the development of hemorrhagic complications, hypochromia may occur due to iron deficiency. The number of peripheral blood leukocytes varies widely (from 1 • 109 / l to 200 • 109 / l), but more often it remains at the subleukemic level and does not exceed 20-30 • 109 / l.

The most pronounced leukocytosis is observed in patients with T-ALL and acute monoblastic leukemia. When calculating the leukocyte formula in 90% of patients with acute leukemia blast cells are detected, the number of which can vary from 1-2 to 100%. In typical cases, there are no intermediate forms of neutrophilic cells between the blasts and mature granulocytes (“leukemic failure”, or hiatus leukaemicus).

In 20% of patients, the number of blast cells exceeds 50 × 109 / l, and in 10% of the blasts in the peripheral blood are absent (pancytopenia and relative lymphocytosis are usually noted). At the level of leukocytes above 100 • 109 / l, the risk of the development of leukostatic complications (neurological disorders, acute respiratory distress syndrome, in men, in addition, priapism) sharply increases.

Thrombocytopenia is detected in the overwhelming number of patients with acute leukemia and is more pronounced in acute myeloblastic leukemia (AML) (in half of the patients the number of platelets is less than 50 • 109 / l). At the same time, in 1–2% of patients thrombocytosis is noted (more than 400 • 109 / l).

In some patients, an increase in prothrombin and partial thromboplastin time may be noted; in acute promyelocytic leukemia, a decrease in fibrinogen level and other signs of DIC are often observed. It should be noted that the development of the syndrome of DIC is possible with any variant of acute leukemia.

Myelogram with acute leukemia. The study of bone marrow aspirate is needed to diagnose and establish a variant of acute leukemia. The number of myelokaryocytes is usually increased, megakaryocytes are absent or their number is reduced. When calculating the myelogram, at least 20% of blasts are detected, constriction of normal hemopoiesis sprouts. To verify the variant of acute leukemia, it is necessary to conduct cytochemical, immunological and cytogenetic studies, the results of which have prognostic significance and allow planning medical tactics.

Infectious complications

Infectious complications (respiratory infections, bacterial sepsis) rarely develop in the onset of the disease, but have important practical and prognostic significance. Complications of various etiologies (bacterial, viral, fungal) usually occur during treatment, most often with the development of agranulocytosis and are one of the main causes of deaths.

Neuroleukemia in most cases manifests with a meningeal symptom complex or a picture of an intracerebral tumor (nausea, vomiting, headache), isolated lesion of cranial nerves is much less common (nn oculomotorius, facialis).

Clinical signs do not allow to reliably differentiate acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL) without performing cytochemical and immunological studies of blast cells. However, some symptoms are more likely to occur in certain types of acute leukemia (OL).

Acute myeloid leukemias are characterized by more vivid symptoms: patients usually have intoxication, anemic, and hemorrhagic syndromes, often with severe infectious complications. 90% of patients with ALI develop syndrome of DIC. Acute monocytic, rarely myelomonocytic leukemia more often than other variants of AML are accompanied by hypertrophic gingivitis, lymphadenopathy, the appearance of leukemides on the skin and high leukocytosis.

In acute erythromyelosis, the anemic syndrome, macrocytosis and peripheral blood normocytosis are leading. Acute megakaryoblastic leukemia is usually characterized by pronounced myelofibrosis, which makes it difficult to obtain bone marrow aspirate and serves as a basis for performing bone marrow trephine biopsy to verify the diagnosis.

Acute lymphoblastic leukemias are characterized by more scarce clinical manifestations: intoxication, anemic and hemorrhagic syndromes are less pronounced, and infectious complications occur less frequently. At the same time, in patients with acute lymphoblastic leukemia (ALL), ossalgia, lymphadenopathy (including an increase in mediastinum), hepatosplenomegaly and neuroleukemia are significantly more common.

Debut of acute leukemia

The debut of acute leukemia has no characteristic clinical manifestations.
For children and young people, a more acute onset is more typical, in which patients are worried about progressive general weakness, malaise, headache, shortness of breath, myalgia, ossalgia, less often – abdominal pain, fever. In mature and advanced age, the disease usually develops more slowly and is characterized by a predominance of symptoms of anemia and intoxication.

More rarely, the first manifestation of acute leukemia (OL) are hemorrhages in the skin, nose bleeds, blood admixture during defecation, mucosal bleeding, hypertrophic gingivitis, and necrotic stomatitis. Finally, in 5% of patients, a diagnosis is made by examining peripheral blood for other diseases.

On examination, most patients determine the paleness of the skin and mucous membranes due to anemia, and manifestations of hemorrhagic syndrome (petechiae and / or ecchymosis on the mucous membranes of the oral cavity and on the skin, signs of internal bleeding are possible). Rarely marked increase in the liver and spleen, lymphadenopathy.

With the developed clinical picture of acute leukemia (OL), the following syndromes are expressed to varying degrees:

1) syndrome of tumor intoxication (weakness, fatigue, weight loss, increase in body temperature, not associated with infection);
2) anemic syndrome (shortness of breath during normal exercise, dizziness, pallor of the skin and mucous membranes);
3) hemorrhagic syndrome (hemorrhage, bleeding mucous membranes, in severe cases – profuse bleeding of various localization, bleeding in the brain);
4) syndrome of leukemic proliferation (ossalgia, swollen lymph nodes, liver, spleen, hypertrophic gingivitis, neuroleukemia);
5) syndrome of infectious complications (local or generalized infections).

Basic data of clinical, laboratory and instrumental studies

In most patients, the symptoms of acute leukemia are not specific, however, the diagnosis is usually not difficult to study the history of the disease, physical data, the study of peripheral blood and myelogram.

I. Symptoms of acute leukemia: Weakness, indisposition, shortness of breath; hemorrhagic manifestations from minimal effects and injuries; weight loss; ossalgia; abdominal pain (rare); neurological symptoms (rare).

Ii. Clinical signs of acute leukemia: Anemic syndrome (weakness, pallor, tachycardia); hemorrhagic syndrome (ecchymosis, petechiae, internal and external bleeding); fever and infections (pneumonia, sepsis, pararectal abscess); lymphadenopathy, hepatosplenomegaly, expansion of the mediastinum; infiltration of the gums and skin (rarely); increased kidney and development of renal failure (rare); damage to the meninges and cranial nerves (rare).

III. Important laboratory and diagnostic tests for acute leukemia. A blood test with leukocyte count; coagulogram with determination of fibrinogen level; biochemical parameters (transaminases, creatinine, uric acid, calcium, phosphorus, electrolytes); a study of aspirate and trepanobioptata (not in all cases) of the bone marrow; determination of the blast cell phenotype, cytogenetic (according to indications – molecular genetic) studies; analysis of cerebrospinal fluid; X-ray of the chest, ultrasound of the abdominal cavity, computed tomography of the chest and abdominal cavity (according to indications in acute lymphoblastic leukemia); HLA-typing (in young patients).

The most significant moments of pathogenesis

1. In most cases, acute leukemia (OL) develops from cells committed in the direction of myeloid or lymphopoiesis, fewer cell lines are involved. This explains the diversity of the clinical course and response to therapy for different types of blast cells that make up the tumor clone.

2. For acute leukemia, tumor progression is characteristic: as the disease progresses, clones of leukemic cells with new properties (morphological, cytochemical, immunological, etc.) appear, which explains the development of resistance to previously effective treatment.

3. The development of most clinical manifestations and laboratory data (anemia, hemorrhagic syndrome, fever) is due to the “crowding out” of normal hemopoietic tissue with a leukemic clone.

4. As the acute leukemia progresses (more rarely, from the onset of the disease), blast cells metastasize beyond the blood-forming organs. This leads to the development of specific (blast) infiltration of internal organs, lymphatic tissue, skin, mucous membranes, and may be accompanied by functional failure of various internal organs, organomegaly, lymphadenopathy, hyperplastic gingivitis, skin leukemides, etc. In some cases, acute leukemia (often – acute lymphoblastic leukemia (ALL)) blast cells metastasize to the central nervous system, which leads to the development of neuroleukemia.

5. The proliferation of blast cells and their death are accompanied by the development of intoxication syndrome and metabolic disorders that occur in most patients.

Mechanisms of development of acute leukemia – pathogenesis

Acute leukemias result from the clonal neoplastic proliferation of blast cells, which are characterized by blockade of differentiation into more mature blood cells and the ability for virtually unlimited division. In acute leukemia, the tumor is a clone – the offspring of a single malignant cell. Accumulating in the bone marrow, leukemic blasts displace normal hematopoiesis cells, which ultimately leads to symptoms of the disease.

In laboratory studies of acute leukemia, about 1011 blast cells are found in the patient’s body, with the development of clinical symptoms – 1012 blasts (approximately one kilogram).

Leukemia cells circulate in the blood and can cause damage to other organs and tissues (the frequency and nature of the lesion depends on the variant of acute leukemia). Unlike chronic leukemias, which have the phenotypic and biological characteristics of more mature cells, acute leukemia develops and is biologically similar to primitive hematopoietic progenitor cells. The phenotypic heterogeneity of leukemic cells suggests that acute leukemia (AL) can occur at various stages of differentiation.