Differential diagnostics

Differential diagnosis of M2 for acute non-lymphoblastic leukemia is performed with myeloblastic without maturation, myelomonoblastic and basophilic-cellular variants. In the M2 variant, in contrast to M1, the number of granulocyte maturation cells is more than 10%. In contrast to variant M4, when M2 there are no monoblasts containing nonspecific esterase. In basophilic cell leukemia, unlike myeloid blast, metachromasia is determined in blasts and peroxidase is absent, the serum of patients with elevated heparin and serotonin levels.

Of particular practical importance is the identification of promyelocytic leukemia among patients with acute non-lymphoblastic leukemia, since the treatment for this variant, including ATRA, is fundamentally different from the treatment regimens used for the remaining myeloid leukemia. The leading diagnostic feature of the M3 blasts is the characteristic dvulobolny form of the nuclei. However, the number of such cells in the blast population may be less than 25%, which significantly complicates the differential diagnosis of variants of MH and M2. Only a careful morphological analysis allows to identify characteristic cells. The lack of grit and Auer rods in the M3v variant makes leukemic promyelocytes similar to maturation monoblasts.

The differential diagnosis of M3v and M5b acute non-imphobular leukemia is based on the high activity of granulocyte enzymes in the first case and non-specific esterase in the second.

Diagnosing a rare variant of APL with t (11; 17) (PLZF / RARa), resistant to ATRA, with powerful cells similar to myeloblasts, presents great difficulties for morphologists. Practically, the diagnosis of this variant is based on data from a cytogenetic study. In the diagnosis of myelomonoblastic acute non-lymphoblastic leukemia, the definition of biclonal (most frequent) and biphenotypic variants is necessary. The visual difference between them is determined by carrying out successive reactions on a single smear of the bone marrow, first with ASD chloroacetate and then with α-naphthylacetate.

In the biclonal variant, 2 types of positive cells are detected: ASD-chloroacetate and α-naphthylacetate. In the bifenotypical variant, granules of both enzymes, colored in different (blue and red) colors, are found in all blasts. An increase in the number of atypical eosinophils suggests that the 16th chromosome is abnormal.

Differentiate M4 from options M2 and M5 according to cytochemical studies and the determination of the content of monocytoid cells.

Modern differential diagnosis of acute leukemia variants

The polymorphism of leukemic elements in acute leukemia is pronounced significantly. The presented schematic description of the signs of cells of different types does not include the whole variety of forms, therefore the differential diagnosis of disease variants can cause certain difficulties. Differences in the diagnosis of three morphologists in the study of the same observations can be 5-10%.

One of the most difficult diagnostic options for acute non-lymphoblastic leukemia is myeloblastic with minimal differentiation. It was singled out by the authors of the FAB classification among the latter. This variant of leukemia has to be differentiated from acute lymphoblastic, megakaryoblastic, bi-phenotypic, and true erythroid leukemias.

The difference between M0 and acute lymphoblastic leukemia and large cell lymphoma in the leukemization stage lies in the expression of myeloid (CD33, CD13) and the absence of lymphoid antigens. PAS-positive substance is more often defined in diffuse, but not in granular form.

In the differential diagnosis of MO variants from M6 and M7, the criterion is the absence of specific immunological markers of the erythroid (GlyA) and megakaryocytic (CD41, CD61) series. In contrast to biphenotypic leukemia, only myeloid antigens are detected on blasts and lymphoid antigens are absent.

In myeloblastic leukemia without maturation, two types of blasts can occur. In blasts of type I, a large nucleol is determined in the nucleus, primary granules are absent, the nuclear-cytoplasmic ratio is high. For blasts of type II, small nucleols are characteristic, the presence in the cytoplasm of several primary granules, a moderate nuclear-cytoplasmic ratio. Acute myeloid leukemia with type I blasts with a low peroxidase content (less than 10%) is differentiated from acute lymphoblaqueosis. In these cases, the data of immunophenotypic research, namely the expression of myeloid antigens CD33 and CD13, are very significant.

In type II myeloblasts, differential diagnosis is performed with the M2 variant. The presence in the bone marrow of less than 10% of ripening granulocytes gives grounds to establish the variant Ml, and not M2 of acute non-lymphoblastic leukemia.

Myeloblastic leukemia with maturation is heterogeneous in its characteristics. Blasts vary in size, number of forms with granularity, degree of disgranulopoiesis, cytogenetic parameters. Comparison of M2 patients with acute non-lymphoblastic leukemia with t (8; 21) and expression of the AML / ETO gene and the absence of this anomaly showed that in the former, compared to the latter in the bone marrow, Auer’s rods were more common, the peroxidase activity was higher, trophilia was determined by pink staining, the number of eosinophils was slightly increased (2–6%).

It should be noted that, on the basis of morphological parameters without data from the karyotype study, it is difficult to distinguish these groups of patients. The clinical course of leukemia with this translocation proceeded in patients more favorably than in its absence.

Algorithm for the diagnosis of acute leukemia

As noted, the modern algorithm for diagnosing variants of acute leukemia includes morphological, cytochemical, immunological and cytogenetic, and sometimes molecular-biological approaches. At the same time, it should be emphasized that the morphological assessment of the composition of punctate is basic in the diagnosis of acute leukemia.

Without counting the myelogram and analysis of the morphological and cytochemical characteristics of cells of the leukemic substrate, it is impossible to interpret the data of immunological and cytogenetic studies.

The diagnostic value of each method is different for different options. Thus, by morphocytochemical methods, most variants (up to 90%) of M1 — M5 acute non-lymphoblastic leukemia can be characterized. However, M0, M7 and some cases of M6 are diagnosed only by an immunological method, as well as acute lymphoblastic leukemia.

The use of immunophenotyping for the diagnosis of acute leukemia using the ICA panel allows determining the linear orientation and / or stage of differentiation of blasts starting from the level of stem progenitors to mature forms. It is produced using flow analyzers or on bone marrow smears by the peroxidase-antiperoxidase (PAP) or phosphatase-antiphosphatase (APAP) method.

Each method has its advantages. Using a flow analyzer provides data on tens of thousands of cells in the leukemic population, the study of cells on smears makes it possible to combine immunological research with morphological and cytochemical.

The widely accepted immunological approach broadens our understanding of the biology of blast cells in acute leukemia. The emergence of new ICA allows you to allocate additional subtypes of blasts. In particular, the use of a new antimonocyte-macrophage ICA D11 (CD68) made it possible to detect myeloid differentiation in cells that were previously regarded as undifferentiated. The data obtained in the study of the mechanisms of apoptosis, played an important role in the study of the prognosis of acute lymphocytic leukemia.

There is a point of view that the variant of leukemia can be established only on the basis of immunological criteria, without using morphocytochemical data. At present, this approach has not found support, since only ICA to myeloperoxydase and lysozyme have defining specificity, and all other antigens are not so specific.

Molecular biological methods are becoming increasingly important in studying the pathogenetic mechanisms of acute leukemia. A number of observations are already used in practical medicine for the diagnosis of variants of acute leukemia. The study of the rearrangement of immunoglobulin genes and T-cell receptors plays an important role in the differential diagnosis of lymphoproliferative diseases.

The characterization of karyotype abnormalities in acute promyelocytic and myelomonoblastic leukemia with eosinophilia, taking into account the data of cytogenetic studies and the PCR reaction, makes it possible to establish a sub variant of leukemia and determine the prognosis of the disease.

Acute lymphoblastic leukemia (ALL) – diagnosis

Acute lymphoblastic leukemia (ALL) includes leukemias, the tumor substrate of which is represented by progenitor cells of the lymphoid series. There are two variants of leukemia, depending on the direction of differentiation of blast cells: acute B-lymphoblastic leukemia / lymphoma from progenitor cells and acute T-lymphoblastic leukemia / lymphoma from progenitor cells.

Most of the observations are regarded as acute leukemia, however, cases with a pronounced extramedullary lesion and a number of blasts in the bone marrow of less than 25%, according to the WHO classification, belong to lymphomas.

The presence of individual cytogenetic abnormalities in acute lymphoblastic leukemia (ALL) has significant prognostic significance, therefore, in B-acute lymphoblastic leukemia (ALL), a number of variants are distinguished depending on the characteristics of the karyotype.
1. B-lymphoblastic leukemia / precursor lymphoma: t (9; 22) (q34; qll); BCR / ABL; t (v; llq23); MLL restructuring; t (l; 19) (q23; pl3) PBX / E2A; t (12; 21) (pl2; q22) TEL / AMN; ALL hypodiploid; ALL hyperdiploid (> 50).
2. T-lymphoblastic leukemia / precursor lymphoma.

The allocation of morphological types of lymphoblasts according to the FAB classification depending on their size [prevalence of microform L1 or mesoform P2] did not play a significant role in determining treatment tactics or in the prognosis of the disease. The type of blasts with basophilia and vacuolation of LZ cytoplasm is more characteristic of Burkitt’s lymphoma and is not specific for progenitor cells. The morphocytochemical signs of B- and T-lymphoblasts have some differences: • B-lymphoblasts are characterized by significant morphological diversity. Cells can be regular or elongated, sometimes they resemble the shape of a hand mirror. The size of the blasts varies from micro to mesoform. The nuclei are round, oval or folded, with dense chromatin and rare nucleoli. The cytoplasm is scanty, slightly basophilic, sometimes contains a slight granularity. The nuclear cytoplasmic ratio is high or moderate.

• B-lymphoblasts do not contain peroxidases, lipids and ASD-chloroacetate esterase. In half of the cases, small granules of PAS-positive substance and acid phosphatase in a fine granular form are found in them.

• T-lymphoblasts are usually monomorphic, medium-sized, rounded, with irregularly folded nuclei. The cytoplasm is scanty, moderately basophilic. The nuclear cytoplasmic ratio is high.

• T-lymphoblasts do not contain peroxidase, ASD-chloroacetate esterase and lipids. PAS-positive substance is rarely found in the form of a single granule. A characteristic feature is the presence of large granules of enzymes: acid phosphatase, nonspecific esterase, and acid nonspecific esterase.

Depending on the stage of lymphoblasts differentiation, subvariants of B-and T-linear acute lymphoblastic leukemia (ALL) are distinguished. The diagnosis is based on cell immunophenotyping data. The frequency of detection of these subvariants is different. Different chromosomal abnormalities are characteristic of different subvariants.

Leukemia from B-linear progenitor cells occurs in approximately 75% of adult patients with acute lymphoblastic leukemia (ALL). In all cases, blast cells express CD19 and / or CD79a and / or cytoplasmic CD22 molecules. As differentiation to these antigens are added, new, specific to the subsequent stages of maturation. Among B-linear leukemias, the following immunosupportants are distinguished: pro-B, pre-pre-B, pre-B, and B-ALL. The presence of t (9; 22), t (4; ll) and t (l; 19), observed in pro-B and pre-pre-B variants, is an independent indicator of an unfavorable prognosis.

Translocation (9; 22) occurs in adult patients with acute lymphoblastic leukemia (ALL) quite often (about 25%). Ph-positive and Ph-negative lymphoblasts do not have morphocytochemical and immunophenotypic differences, therefore the data of cytogenetic studies are necessary when choosing the tactics of treatment in patients with acute lymphoblastic leukemia (ALL). The prognostic value for subvariants of B-linear acute lymphoblastic leukemia (ALL) also has a change in the number of chromosomes in the blasts: hyperploidy is favorable, and hypoploidy is an unfavorable sign.

T-linear variants occur in approximately 25% of cases of acute lymphoblastic leukemia (ALL). Two immunosuppressants are distinguished: early pre-T and mature T-ALL. Both of them are characterized by the expression of the CD7 antigen. Pre-T-acute lymphoblastic leukemia (ALL), in turn, is subdivided according to the presence of the “common” antigen into CD10-positive and CD10-negative subvariants. T-linear leukemias are characterized by rearrangements of T-cell receptor genes located on the 14th and 7th chromosomes, in particular t (10; 14), t (ll; 14), t (l; 14) and inv14.

Acute leukemia of unclear differentiation line – diagnosis

A special group is made up of acute leukemias, in which the cells simultaneously exhibit a myeloid and lymphoid orientation of differentiation. In this case, two subclones of cells can be determined, each of which expresses markers characteristic of a single line. These leukemias are called biclonal. In other cases, the blasts coexpress both myeloid and lymphoid or B-or T-lymphoid antigens. This variant of the disease is called biphenotypic acute leukemia (BAL).

Myeloid differentiation is determined by the presence of peroxidase, esterase and / or expression of myeloid (CD13, CD33 and CD65) antigens in blasts. B-lymphoid differentiation is detected by expression of the antigens CD79a, cytoplasmic CD22, cytoplasmic IgM, CD19, CD10 and CD20. T-lymphoblasts are characterized by reactions with MCA to CD3, TCR, CD2, CD5, CD8.

In order to make a diagnosis of biphenotypic leukemia, it is necessary that a positive result be observed with at least two ICA of the same line. It should be noted that some antigens (T-linear CD7, CDla and B-linear CD24) are often present on myeloid cells and cannot serve as markers of the bifenotype. Due to the difficulties in interpreting the results, it became necessary to unify the diagnosis of hybrid leukemia. According to the BAL system, acute leukemia is considered biphenotypic if the sum of cells with myeloid and lymphoid markers exceeds 120. The presence of mixed differentiation is sometimes found only at the molecular-biological level.

It has been shown that during ONLL, rearrangements of the genes of immunoglobulin chains or T-cell receptor genes can be detected.

Biphenotypic leukemias are characterized by a high frequency of cytogenetic abnormalities. In a third of cases of B-lymphoid / myeloid proliferation, the Philadelphia chromosome is determined by t (9; 22), in separate cases — 1 (4; 11) (q21; q23) and anomalies llq23. In patients with T-lymphoid / myeloid biphenotypic leukemia, complex abnormalities of the karyotype are often found.

Acute leukemias of unclear differentiation line

The category of such leukemias include acute leukemias, in which neither morphological, nor cytochemical, nor immunophenotypic methods allow to establish the differentiation of blasts along any line of hemopoiesis (acute undifferentiated leukemia), as well as observations when morphological and / or immunophenotypic data indicate the presence signs of maturation along two lines of hemopoiesis: myeloid and lymphoid or B- and T-lymphoid (acute bilinear and acute bifenotypic leukemia).

In acute undifferentiated leukemia, the morphological and cytochemical indicators of the blasts do not have any characteristic features. Cells react only with µa to early hematopoiesis precursors – stem-cell CD34, contain the enzyme deoxynucleotidyltransferase (TdT), and also express the antigens CD38 and histocompatibility class II HLA-DR.

The First European Group on the Immunological Classification of Leukemia (EGIL) recommended that further studies of this undifferentiated variant be conducted to prove the myeloid or lymphoid pattern of cell differentiation. It is proposed to use a wide panel of 33 immunological markers. According to the authors, the study of blast cells should be carried out in 2 stages. First, it is necessary to study the linear non-specific antigens TdT, CD34, HLA-DR and establish B, T and myeloid linear directivity. Then it is necessary to determine the stage of differentiation of leukemic cells. To clarify the diagnosis, it is proposed to investigate the rearrangement of immunoglobulin genes and T-cell receptors.

Cases of undifferentiated acute leukemia are sometimes referred to as stem-cell. It has been established that the group of patients with such leukemia is heterogeneous according to the blasts phenotype. The following immunodvariants of this acute leukemia are distinguished: 1. CD34 +, HLA-DR-, CD38-; 2. CD34 +, HLA-DR +, CD38 ~; 3. CD34 +, HLA-DR \ CD38 +.

Acute non-lymphoblastic leukemia due to therapy – diagnosis

Acute non-lymphoblastic leukemia and myelodysplastic syndrome can be caused by two types of therapy: either using alkylating agents and / or radiation therapy, or type II topoisomerase inhibitors. In the latter case, lymphoid leukemias can also be observed.

After therapy with alkylating agents and radiation, acute non-lymphoblastic leukemia occurs after 5 years or more. The risk of acute non-lymphoblastic leukemia depends on the total cumulative dose of the drugs, the age of the patient, and the combined use of the two methods of treatment. The onset of the disease is manifested in the form of myelodysplastic syndrome with cytopenia, multiline dysplasia.

Dysgranulo- and dyseritropoiesis are observed in the majority of patients, the number of basophils is increased. Often (up to 60% of cases) ring sideroblasts are found, in neutrophils, peroxidase deficiency is determined. As a rule, there is myeloblastic acute non-lymphoblastic leukemia, but there can also be myelomonoblastic, erythroblastic, megakaryoblastic and, less commonly, promyelocytic variants. The immunophenotype of blasts is characterized by heterogeneity.

They express myeloid antigens (CD13, CD33), early CD34, CD56 and / or lymphoid CD7. In the karyotype, unbalanced translocations or deletions of the 5th and / or 7th chromosome are often detected with the loss of all or part of the long arm of the chromosome. There are anomalies of other chromosomes: 1st, 12th, 14th, 18th, complex chromosomal aberrations.

Acute leukemia after therapy with topoisomerase II inhibitors occurs faster than after the use of alkylating agents. The latent period is on average 33-334 months. Among the drugs distinguish epipodophyllotoxins (etoposide and teniposide) and anthracyclines. Acute leukemias caused by these drugs, as a rule, belong to monoblastic or myelomonoblastic, although other variants of acute nonlymphoblastic leukemia, as well as acute lymphoblastic leukemia can occur. The most frequent violation of the karyotype is the presence of the llq23 abnormality as a result of translocations (9; 11), (11; 19), (6; 11).

In addition, translocations can be detected (8; 21), (3; 21), (6; 9). ALL is usually associated with t (4; ll) (q21; q23). In secondary acute leukemia, remission is less frequent and shorter than in primary.

Acute non-lymphoblastic leukemia with recurring cytogenetic abnormalities

Characteristic cytogenetic abnormalities in acute non-lymphoblastic leukemia (ONLL) are usually represented by balanced translocations. Such recurring, or non-random, cytogenetic anomalies include t (8; 21), t (15; 17), t (l6; 16), inv (16) and an anomaly llq23, resulting from the translocation of chromosome 11 with various partners (about 20) [61]. For most of these leukemias, a high frequency of complete remissions and a more favorable prognosis are typical. Some morphological features are characteristic of them.

Acute myeloid leukemia with t (8; 21) (q22; q22); (AML1 / ETO). This anomaly is detected in 5-12% of cases of acute non-lymphoblastic leukemia (ONLL), more often in young patients, occurs predominantly with myeloblastic leukemia with maturation (M2 according to FAB classification), sometimes with myeloblastic leukemia without maturation (Ml) or myelomonoblastic (M4).

Blasts express both myeloid (CD13, CD33), and often the lymphoid antigen CD 19. CD34 and CD56 are less commonly defined. For elements of the neutrophilic series, marked dysplasia, the presence of pelgheroid forms, homogeneous pink staining of the cytoplasm are characteristic. In mature neutrophils, in some cases Auer rods can be detected. The number of eosinophils may be increased, but there is no dysplasia in them. The presence of t (8; 21) gives a basis, when the number of blasts is less than 20%, to diagnose ONLL, and not MDS.

In ONLT with t (8; 21), the prognosis is good, with a high frequency of complete remissions and a long duration of survival with adequate treatment. Expression of the CD56 antigen worsens the prognosis.

Acute myeloid leukemia with inv (16) (pl3; q22) or t (16; 16) (pl3; q22); (CBFb / MYH11). In this case, leukemia (M4eo according to the FAB classification) in the bone marrow myeloblasts, monoblasts and anomalous eosinophil component are determined. Immunophenotyping on blasts reveals common myeloid antigens (CD13, CD33, MPO) and markers of monocytic differentiation (CD14, CD11b, CD11c, CD64, CD36, and also lysozyme). Promyelocytes and myelocytes of the eosinophilic series are distinguished by the presence of pathological granularity.

Determining the type of blast cells in acute leukemia

The study of α-naphthyl acetaserase plays a major role in the diagnosis of monoblasts M5a and M5b. This enzyme is found in most hematopoietic cells, but its isoform, inhibited by sodium fluoride, is characteristic only of cells of the monocyte-macrophage line. In determining non-specific esterase, various substrates are used with equal success: a-naphthyl acetate, butyrate, AS-chloroacetate, etc. Their diagnostic value is close.

The activity of the enzyme in monoblasts without maturation and with maturation, as a rule, is equally high. At the same time, using a panel of various substrates for nonspecific esterase, it is possible to carry out a presumptive differential diagnosis of monoblast and megacaryoblastic leukemias before immunophenotyping.

The result of the PAS reaction has a certain diagnostic value, since a characteristic of most myeloid blasts is the diffuse arrangement of the PAS-positive material in contrast to the lymphoblasts, which are characterized by the arrangement of the reaction product in the form of granules. The results of this study help to distinguish between MO ONLL and ALL. In some cases, in myeloblasts of type MO and erythroblasts, the PAS-positive substance may be in the form of granules.

Different types of blast cells in acute non-lymphoblastic leukemia (ONLL) differ in the antigenic structure of their membranes. The expression of antigens CD33, CD13 is characteristic of the cells of the granulocyte line, they react with ICA to peroxidase. The monocytic line blasts interact with the ICA for lysozyme. The presence of glycophorin A molecules is characteristic of erythroid cells, and CD41 and CD61 are characteristic of megakaryocytes.

Immunological data do not allow a differential diagnosis of granulocytic line blasts, however, the use of immunophenotyping data is crucial for the diagnosis of M0, M6 and M7 variants, since the morphological and cytochemical indicators of these cells do not allow to establish the type of blasts.

Mielosarcoma – morphology, diagnosis

Melosarcoma. This form is included in the WHO classification in the section of acute non-lymphoblastic leukemia (ONLL):

• extramedullary tumor from blast or maturing myeloid cells in patients with various myeloproliferative diseases or myelodysplastic syndromes;
• the presence of myelosarcoma in patients with acute non-lymphoblastic leukemia (ONLL) may be the first manifestation of the disease or a relapse of ONLL during remission. In patients with myeloproliferative diseases, it is associated with the onset of a blast crisis;
• blasts are more often represented by myeloblasts, but they can be cells of any myeloid line;
• type of blasts is determined on the basis of morphocytochemical and immunological studies;
• myeloblasts with maturation can have a translocation (8; 21), myelomonoblasts with eosinophils – inv16 or t (16; l6), monoblasts – anomaly 11q23.

The determination of the type of blast cells and, accordingly, the variant of acute non-lymphoblastic leukemia (ONLL) is based on the complex characteristics of the morphological, cytochemical and immunophenotypical features of leukemic elements.

The morphological criteria for the characteristics of blasts include cell size (ratio of macro-, meso- and microforms), the shape of nuclei (round, folded, monocytoid), the presence of grain and / or Auer rods, nuclear-cytoplasmic ratio (high, moderate or low). It is on the basis of morphological signs that leukemic myeloblasts and monoblasts are divided into cells with the presence or absence of signs of maturation.

Cytochemical markers of granulocyte blasts are peroxidase, lipids detected by Sudan black B, and ASD-chloroacetate esterase. The content of these markers in myeloblasts varies considerably, sometimes only one of them is detected. In the case of a dubious response, it is imperative to carry out two cytochemical reactions – peroxidase and lipids, in order to avoid a possible error.

The activity of ASD-chloroacetate esterase is significantly lower than peroxidase, therefore the definition of this enzyme is of lower diagnostic value. Low peroxidase activity in myeloblasts is a poor prognostic sign.