OBJECTIVE:To evaluate and compare morphometric and histotopographic characteristics of megakaryocytic lineage in preparations stained with H&E or antibodies to CD42b in diagnostic trepanobioptates of bone marrow of patients with primary myelofibrosis and essential thrombocythemia with JAK2 or CALR mutation. Analyze the dimensions and quantity of CD42b-positive megakaryocytes in 1 mm2 area of section and assess suitability of these parameters as an additional differential pathomorphological criterion. MATERIAL AND METHODS:108 trephine biopsies of the bone marrow from patients with primary myelofibrosis (N=53) and essential thrombocythemia (N=55) with JAK2 or CALR mutation were selected. Digitized bone marrow slides stained with H&E or antibodies to CD42b (clone EP409) were the object of study. In every sample the average values of perimeter and area of megakaryocytes were analyzed, as well as the average number of megakaryocytes in 1 mm2 area of myeloid tissue section. Logistic regression analysis was used to describe the relationship between CD42b-positive megakaryocyte characteristics and disease (primary myelofibrosis or essential thrombocythemia). RESULTS:Immunohistochemical examination of bone marrow biopsy specimens using antibodies to CD42b in comparison with H&E staining allows to multiply the number of identifiable megakaryocytes in myeloid tissue by 3.5-4 times (p<0.0001). Statistically significant differences in the mean values of the number of megakaryocytes in 1 mm2 of the section area and megakaryocyte perimeter in patients with primary myelofibrosis and essential thrombocythemia have been demonstrated. ROC analysis (AUC=0.84, 95% CI 0.7782-0.9199) justifies the inclusion of the average perimeter size of CD42b-positive megakaryocytes and their number in 1 mm2 of the section area in the differential diagnostic panel as an additional pathomorphological criterion. CONCLUSION:The revealed statistically significant differences in quantitative and geometric characteristics of megakaryocytes allowed to calculate differential threshold values of characteristics of megakaryocytic lineage of myeloid tissue in diagnostic trepanobioptates of bone marrow from patients with primary myelofibrosis and essential thrombocythemia. Counting the number of CD42b-positive megakaryocytes in one field of view at a magnification of 400 times was proposed as an additional pathomorphological differential-diagnostic sign.

The article reviews the changes in the structure of classification, diagnostic criteria for myeloid and histiocytic neoplasms in the 5th edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues (2022). Information is presented regarding new nosological forms, renaming and abolition of some previously existing ones. The importance of molecular genetic studies in the isolation of myeloid and histiocytic neoplasms and the need to apply these studies in clinical practice are emphasized. Myeloid and histiocytic precancerous and proliferative processes, genetic tumor syndromes, introduced into the classification for the first time, are considered.

Kikuchi-Fujimoto disease (KFD) is a rare disease that is clinically manifested mainly by fever and lymphadenopathy. KFD was originally believed to occur primarily in East Asia women, this disease was subsequently described in all ethnic groups worldwide. The important differential diagnostic feature of KFD is the detection of CD123-expressing plasmocytoid dendritic cells (PDCs) in the tissue of the affected lymph node. The standard immunohistochemical staining method has sufficient sensitivity and specificity to detect CD123, but it gives no way of judging the possible phenotypic heterogeneity of cells with CD123 expression. OBJECTIVE To identify the phenotypic heterogeneity of CD123-expressing cells in the affected lymph nodes in patients with KFD by a sequential immunoperoxidase labeling and erasing (SIMPLE) method. MATERIAL AND METHODS Excision biopsies of lymph nodes were examined in 3 patients with KFD. After an immunohistochemical reaction using a single antibody, the tissue specimen was digitized with a Pannoramic 250 Flash III scanner (3DHISTECH, Hungary), then the cover glass was removed from the section, the specimen was hydrated and placed in a specialized buffer. Then the following primary antibody was applied to the washed tissue specimen and further immunohistochemical reaction and scanning were performed. As a result, each tissue specimen was sequentially stained in reactions with 4 antibodies. The microphotographs of specimens stained in a reaction with anti-CD123 antibody showed positive cells for their identification in the Pannoramic Viewer program (3DHISTECH, Hungary) on the remaining microphotographs displaying the expression of the other 3 markers. The selected fields of view were exported to a JPG format. RESULTS Assessing the co-expression of the antigens CD123, MNDA, CD68, and TCL1A detected 4 CD123+ cell subpopulations: No. 1. CD68+/ MNDA+/ TCL1A+; No. 2. CD68+/ MNDA+/ TCL1A-; No. 3. CD68+/ MNDA-/ TCL1A+; No. 4. CD68-/ MNDA-/ TCL1A+. CONCLUSION SIMPLE has shown the phenotypic heterogeneity of CD123-positive cells (some of them may be PDCs) and could identify 4 immunophenotypically distinct subpopulations in the affected lymph nodes in patients with KFD. Further investigations are needed to define the role of subpopulations in the pathogenesis of KFD and other diseases.

The DBSCAN clustering algorithm, an in-house method of unsupervised machine learning, was used to explore specific histotopographical features of the megakaryocytic lineage in the bone marrow biopsies of patients with JAK2- or CALR-mutated essential thrombocythemia and prefibrotic primary myelofibrosis. Ninety-five bone marrow biopsies of patients with essential thrombocythemia and primary myelofibrosis were investigated to assess the histotopography of megakaryocytes, specifically, the mean number of megakaryocytes in one cluster, as well as the mean number of clusters and megakaryocytes per 1 mm2 of section area. The logistic regression model was statistically significant: χ2 = 14.703, p = 0.023, Nagelkerke R2 = 19.6%. Analysis of the histotopographical features of megakaryocytes allowed correct differentiation between essential thrombocythemia and primary myelofibrosis in 71.6% cases. The differences in the histotopographical features of megakaryocytes in the bone marrow of with JAK2- or CALR-mutated essential thrombocythemia and primary myelofibrosis revealed by the DBSCAN clustering algorithm indicate that a relationship exists between the disease and the pattern of megakaryocytic lineage development that can be used to create a logistic regression model for differentiating these diseases.

The new coronavirus infection is a highly contagious infection caused by the SARS-CoV-2 virus that has become a global public health problem. The pathogenesis of this virus has not yet been clearly understood, the principles of hyperinflammatory immune response in critically ill patients, which leads to acute respiratory distress syndrome and multiple organ failure, innate and adaptive immune responses in the process of structuring the data under study. The interaction of the virus and a macroorganism includes 4 stages: infection, dissemination, cytokine storm, pulmonary fibrosis. This review analyzes the predictors of infection, its possible pathogenesis, the immune response of the macroorganism, as well as the histological characteristics of damage to immune organs; shows receptors for SARS-CoV-2 (ACE2, TMPRSS2) in some organs.

OBJECTIVE:To assess the applicability of imported tissue-marking dyes and the samples of experimental dyes and decorative acrylic paints to mark the resection margins of a surgical specimen.MATERIAL AND METHODS:Three sets of tissue-marking dyes: 2 imported sets and 1 experimental set, each containing red, orange, yellow, green, blue, purple, and black dyes, and a set of decorative acrylic paints containing black, blue, light blue, green, yellow, ocher, orange, magenta, and purple dyes. The experimental dyes and imported ones were used to mark tonsillar fragments obtained at tonsillectomy. The set of experimental dyes and that of decorative acrylic paints were used to stain the fragments of autopsy specimens (the skeletal muscles, pancreas, and large bowel). The tissues obtained at autopsy were marked before and after fixation in 40% formalin for 30 min and 24 hours. The specimens were subjected to standard tissue processing. Paraffin blocks were cut into 5-μm sections that were stained with hematoxylin and eosin. To estimate resection margin marking, each specimen was examined by 7 researchers who independently assessed the covering ability of a dye and its color in the paraffin block and microslides.RESULTS:All researchers correctly identified purple, black, and green colors from the three sets of dyes in the surgical tonsillar specimen. When examining the autopsy specimens, all the experts correctly recognized magenta and green decorative acrylic paints and black and blue experimental dyes. The time of fixation and the type of tissue did not affect the color of a dye in the paraffin block and tissue specimen.CONCLUSION:Some of experimental dyes and decorative acrylic paints are highly competitive with imported tissue-marking dyes in their characteristics, are correctly recognized in the block and tissue specimen under a microscope, and can be proposed to mark the resection margins of the examined tissues.

Aim. To study the relationship of karyotype, JAK2, CALR, and MPL driver mutations and ASXL1 mutation status with the progression and prediction of primary myelofibrosis (PMF). Materials & Methods. The trial included 110 PMF patients (38 men and 72 women), median age was 59 years (range 18-82) with median follow-up after diagnosis of 2.6 years (range 0.1-23). The patients were examined for JAK2, CALR, MPL, and ASXL1 mutations. Restriction fragment length polymorphism technique was used for the analysis of V617F substitution in JAK2 and 515 codon mutation in MPL. CALR (exon 9) and ASXL1 (exon 12) mutation tests were performed using Sanger direct sequencing. In 48 (44 %) out of 110 patients bone marrow cell karyotype was determined. Clinical and hematological parameters and median overall survival (OS) of patients were analyzed with regard to detected genetic aberrations and combinations of them. Results. JAK2, CALR, MPL mutations were detected in 55 (50 %), 28 (25.5 %), and 7 (6.4 %) out of 110 patients, respectively. Triple negative (TN) status was identified in 20 (18.2 %) out of 110 examined patients. ASXL1 mutations were detected in 22 (20 %) out of 110 patients. Out of 48 patients in 32 (66.7 %) normal karyotype, in 3 (6.3 %) favorable karyotype, in 4 (8.3 %) intermediate-prognosis karyotype, and in 9 (18.7 %) unfavorable karyotype were detected. The comparison of clinical and hematological parameters showed a number of significant differences. JAK2-positive patients had a higher hemoglobin level (median 129 g/L; p = 0.021). TN was associated with a high IPSS risk (p = 0.011), low hemoglobin level (median 101 g/L; p = 0.006), drop in platelet count (median 266 x 109/L; p = 0.041), increased lymphocyte count (median 26.9 х 109/L; р = 0.001). The detection of terminating mutations in ASXL1 correlated with palpable enlarged spleen (р = 0.050), reduced platelet count (median 184 х 109/L; р = 0.016), leukocyte count > 25 х 109/L (р = 0.046), and blast count > 1 % (р < 0.001). Univariate regression analysis showed that terminating mutations in ASXL1 (hazard ratio [HR] 2.9; р = 0.018), unfavorable karyotype (HR 8.2; р < 0.001), and TN (ОР 8.1; р < 0.001) had prognostic value for OS. ASXL1 mutation was associated with significantly worse OS in TN patients. Median OS of ASXL1-negative patients without high-risk chromosomal aberrations was significantly longer than in patients with high-risk karyotype and/ or ASXL1 mutation. Conclusion. Several genetic defects in tumor cells are associated with phenotypic manifestations of PMF. Based on the results of cytogenetic analysis and mutation determination of JAK2, CALR, MPL, and ASXL1, patients can be classified in different “genetic” risk groups when PMF is diagnosed.