Method Of Flow Cytofluorometry Research Articles

New prognostic criterion for the outcome of pregnancy in patients with threatening preterm birth.

To identify new criteria for predicting the outcome of pregnancy in women with threatened preterm labor based on the establishment of the characteristics of differentiation and functional activity of memory T-cells in the population of CD8 + T-lymphocytes. The study involved 56 women with threatened preterm labor at 22-34 weeks of pregnancy. Depending on the outcome of pregnancy, patients were divided into 3 groups: Group I included 22 women by premature labor; group II - 34 women whose pregnancy ended by deliver at term. By tricolor flow cytofluorometry method the content of peripheral Tn, Tcm, Tem and Temra cells in CD8+ population was studied. Statistical analysis was carried out using programs «Statistica for Windows 13.0», «Microsoft Excel 2019» and «MedCalc 19.3». Patients with the threat of preterm birth were characterized by a significant increase in the relative content of CD8+ T-lymphocytes in the peripheral blood compared to the control group (p=0.001). When comparing this indicator in the subgroups of women with the threat of preterm birth, it was found that preterm birth was associated with significantly higher relative CD8 + lymphocyte counts (p=0.03) compared to the indicators of the subgroup of women who gave birth on time. When evaluating the results obtained, it was found that the percentage of CD8+Temra GranzymeB-producing cells in the group of patients with threatened preterm birth, whose pregnancy ended in preterm birth, was significantly lower than in the group of women who gave birth on time (p=0.003). The content of Tcm, Tem memory cells producing GranzymeB in the subgroups of women with pregnancy outcomes of timely and preterm birth did not have significant differences compared to the control group. Thus, the prediction of preterm birth is possible with a value of GranzymeB-producing CD8 + Tem equal to 8.2% or less (sensitivity-87.9%, specificity-85%, accuracy-87.9%). Thus, the identification of a new criterion will make it possible to predict preterm birth in a timely manner and promote the choice of optimal pregnancy management tactics, reasonable prevention of fetal respiratory distress syndrome and transfer of a woman to an obstetric hospital designed to provide high-tech care to premature newborns.

Activation of Apoptosis and Autophagy by Gratiola Officinalis Extract in Human Tumor Cell Lines

The problem of creating antitumor drugs with new mechanisms of action that predominantly induce apoptosis is still topical. The extract of Gratiola officinalis is a potential antitumor agent containing mainly flavonoids. The aim of this research is to study the effects of Gratiola officinalis extract on activation of apoptosis and autophagy in breast adenocarcinoma SK-BR-3 and kidney carcinoma A-498 lines. Apoptotic activity of the extract was studied by flow cytofluorometry using Hoechst stain and double staining with annexin V plus propidium iodide. There was 96.3% of cells in SK-BR-3 culture in late apoptosis phase detected by flow cytofluorometry method at the extract concentration of 0.88 mg/ml, 86.3% of cells were in apoptosis by Hoechst stain. The concentration of 0.82 mg/ml caused apoptosis in half of the cells. The extract has cytoprotective activity at low concentration (0.0352 mg/ml). The cytoprotection mechanism is realized through the activation of autophagy. The maximum number of autophagosomes in kidney carcinoma cells is observed at the extract concentration of 0.056 mg/ml. Thus, Gratiola officinalis extract is able to block cytoprotective autophagy with increasing the extract concentration and to activate apoptosis in 85% of tumor cells. Detailed research should be continued to understand the mechanisms of antitumor activity of Gratiola officinalis extract.

Absolute Neutropenia and Infection Development in Premature Infants in Early Neonatal Period: Cross-Sectional Study

Background. Premature infants have high risk of developing of neutropenia and infections in the early neonatal period. The correlation of these events requires further studies.Objective. The aim of the study was to investigate the frequency of absolute neutropenia and infectious complications cases in premature infants in the early neonatal period with estimation of phenotypical and functional features of cord blood neutrophils.Methods. The study included premature infants (gestational age 25–36 weeks) with APGAR score < 8 on the 1st and 5th minutes of life. The frequency of absolute neutropenia (at least once < 1.5109/l) and infectious complications (localized infections of bacterial etiology, early neonatal sepsis) cases in the first 14 day of life was analysed. Additionally, we have determined the expression of CD64, CD16, CD32 by cord blood neutrophils in premature (n = 102) and mature infants (n = 30) via method of flow cytofluorometry. We have used FITC labeled Escherichia coli to estimate their phagocytic activity, and stimulation of E. coli neutrophils in the presence of 5 mM of dihydrorhodamine 123 to estimate their stimulation index (ratio of mean fluorescent intensity (MFI) of activated neutrophils in stimulated samples and in negative controls, E. coli free samples).Results. The episodes of absolute neutropenia in the first 14 days of life were recorded in 17 cases, infectious complications — in 87 children (in 24 cases — sepsis) in the group of premature infants. The frequency of infectious complications in premature children did not correlate with the frequency of absolute neutropenia episodes. Cord blood neutrophils in premature infants had higher CD64 expression and, on the contrary, lower CD16 expression, as well as low phagocytic activity and stimulation index value (in all cases p < 0.001).Conclusion. Absolute neutropenia in premature infants in early neonatal period does not correlate with high risk of bacterial infections. However, cord blood neutrophils in premature infants had lower functional activity.

Comparative DNA cytometry of primary and recurrent soft tissue sarcomas.

e22516 Background: Soft tissue sarcomas (STS) are aggressive tumors with a high degree of recurrence. Radical resection within healthy tissues allows to reduce the recurrence percentage to 25-30% without subsequent therapy. The literary analysis has shown that the study of various biological properties of primary and recurrentsoft tissue tumorsis being conducted. However, currently there is a lack of information to understand the reasons for STS recurrence. The goal of investigation was to reveal the distinctive features of the DNA content and cell distribution in the phases of the cell cycle of recurrent STS. Methods: DNA cytometry in the tumor tissue of 30 primary soft tissue sarcomas and 30 STS recurrences was carried out using the method of flow cytofluorometry. The tumor ploidy and cell distribution in the cell cycle phases were analyzed. Results: A number of differences in the DNA cytometric parameters of primary and recurrent STS have been revealed, they include: an increase in the proportion of aneuploid tumors in case of recurrence, the number of tumors with DNA index within the mitotic cycle, an increase in the proportion of cells in G2+M- phase of diploid and aneuploid tumors and a decrease in S- phase of aneuploid ones. It has been shown that with a G2 differentiation degree, the proportion of cells in G2+M, S- and proliferation index of recurrent STS is significantly lower than the primary parameters. An increase in the proportion of cells in G2+M- phase and a decrease in the rate of proliferation of recurrent STS, depending on the stage, are shown only in case of stage III. Conclusions: The revealed features of DNA content and cell cycle of tumor cells of soft tissue sarcomas will allow to approach to understanding of biological bases of recurrence of this malignant disease.

SPECIFIC INTERACTIONS BETWEEN LECTINS AND RED BLOOD CELLS OF CHORNOBYL CLEANUP WORKERS AS INDICATOR OF SOME LATE RADIATION EFFECTS

Growing interest in lectins is based on their diagnostic and pharmacological potential, especially the ability to inhibit proliferation and initiate apoptosis of cancer cells. In our research microplate lectinoassay able to detect carbohydrate containing structures (receptors) on erythrocyte surface have been proposed for Chornobyl cleanup workers (1986) monitoring. It was expected to reveal specific abnormalities associated with pathological condition arising as a result of late radiation effects. Red blood cell (RBC) specimens were taken from 171persons distributed into the six cohorts: nonexposed donors (1); chronically exposed to the doses below (2) and over 50cGy (3); exposed to acute radiation without (4) and with manifestation of acute radiation syndrome (5and 6). Lectins from 24species of medicinal plants were purified by ethanol fractionation and electrofocusing. Intensity of lectin-receptor interactions was determined in reaction of hemagglutination. Method of flow cytofluorometry was used to study B-cell counts. Hormone levels in blood serum were determined by radioimmunoassay. An elevated ability of RBC to interact with the panel of lectins was found in all cohorts of exposed persons versus nonexposed donors, moreover, changes in the intensity of lectin-receptor binding depended on the dose of irradiation. Diagnostic value of specific RBC reactions with some individual lectins has been elucidated. Elevated intensity of RBC reaction with Zea mays lectin was accompanied by a decrease in serum content of thyroid hormones T4and T3, as well as reduction of B-cell counts. In the case of Rubus caesius lectin the more intensive reaction with RBC, the higher level of hormone cortisol was observed. Deviations from donor's norm in intensity of lectin - RBC interactions in radiation exposed men are supposed to carry information about negative changes in their health status following Chornobyl catastrophe and show the diagnostic potential. The most sensitive reactions have been associated primarily with shifts in endocrine and immune systems. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

Expression of Fc-Gamma Receptors of Neutrophils in Preterm Infants of Different Gestational Age

Background : Premature infants have a high risk of bacterial infections facilitated by the immaturity of their immune system. Objective : Our aim was to study the expression of Fc-gamma receptors of neutrophils and the possibility of its modulation in preterm infants (with a low, LBW, very low, VLBW and extremely low, ELBW, birth weight) with bacterial infection. Methods : We examined cord blood samples, as well as peripheral venous blood samples of newborns taken at 28–30th day of life. The flow cytofluorometry method was used to determine the level of surface expression of Fc-gamma receptors by neutrophils (CD64, CD32, CD16), phagocytic activity and oxygen burst of granulocytes stimulated with fluorescently labelled Escherichia coli. Results: We examined blood samples of 10 full-term infants, 20 preterm infants with LBW, 18 preterm infants with VLBW and ELBW. CD64 expression in preterm infants at birth was higher than in full-term children and made 6.0 (5.3; 9.8), 7.1 (5.1; 11.5), and 4.27 (2.4; 5.8) MFI respectively (p = 0.013). CD16 expression in preterm infants, in contrast, was lower: 99.7 (71.3; 126.0) and 80.9 (58.7; 114.8), and 125.3 (95.5; 144.1) MFI respectively (p = 0.021). By the end of the 1st month of life, CD16 expression of peripheral blood neutrophils in preterm infants increased to 126.6 (110.1; 129.0) MFI in children with LBW and to 118.5 (99.5; 132.2) MFI — in children with VLBW/ELBW. CD32 expression in compared groups did not differ. A correlation between the intensity of the oxygen burst of neutrophils and gestational age was established (r = 0.67; p <0.001). Neutrophil culture of preterm infants in the presence of granulocyte colony stimulating factor (G-CSF) results in a 2.4–5-fold increase in CD16, CD32 expression and enhancement of oxygen burst. Conclusion : Preterm infants are characterized by deregulated expression of Fc-gamma receptors and functional impairment of neutrophils. G-CSF modulates the expression of CD16 and CD32 on the surface of neutrophils and enhances the oxygen burst in them.

Impact of Preoperative Detection of Sodium-Iodide Symporter Expression Level on Differentiated Thyroid Cancer (DTC) Prognosis

Nowadays it is of utmost importance to forecast a cancer progression, in particular, thyroid cancer in order to make a decision about the optimal treatment tactics. Aim. To evaluate the possibility of preoperative detection of membrane located NIS expression level by flow cytofluorometry (FC) method in fine needle aspiration biopsy (FNAB) material as a marker of unfavourable prognosis of DTC. Materials and methods. The research was of prospective character. 43 patients with DTC who underwent medical treatment in general surgery department of St.Petersburg State Medical University named after Pavlov in the period 2009–2010 were enrolled in the study. Level of NIS expression in FNAB material analysed preoperatively and expression was accessed quantitavely by FC method. According to the results of routine histology examination: 28 patients needed radioiodine ablation (RIA). During 48 months of observation recurrences were detected in 10 cases. All recurrences were of local character. Not a single patient from the group without RIA had recurrence. Results. FC method of NIS detection in FNAB material is technically possible and the results obtained are in line with immunohistochemical method. When studying the level of membrane located NIS expression in DTC it was found that the mean level in the group without RIA and disease recurrence is 6.5% with maximum up to 11.6 %. The lowest mean level of NIS expression was in patients group with recurrence of DTC after RIA ( p = 0.00083). We proved that crutial for recurrence of DTC after RIA were decreased level of membrane located NIS expression less than 1%. That means that when NIS level is detected to be less than 1% a patient can be considered to belong to a high-risk group and more aggressive surgical tactics must be used to decrease the risk of recurrence. Conclusion. It is possible to use FC method of detecting NIS expression level on preoperative stage in FNAB material. If it is detected that NIS expression level is lower than 1%, these patients belong to high-risk group and for this group thyroidectomy and central compartment lymph node dissection are recommended.

Clinical and laboratory correlation in patients with consequences of traumatic brain injury

The aim of this study was to establish main peculiarities of apoptosis of peripheral blood leukocytes in patients with traumatic brain injury consequences of varying severity under conditions of disease progression Methods . In 220 patients with the effects of light (71), moderate (59) and hard (90) TBI and the catamnesis of trauma in the age from 1 to 30 years percentage of peripheral blood leukocytes in stage of apoptosis (ANV +) and necrotic (PI +) and the level of reactive oxygen species (ROS +) was explored by flow cytofluorometry method. The control group consisted of 30 people representative by age and sex. Neurological status was assessed by Neurological Outcome Scale for Traumatic Brain Injury (NOS-TBI), cognitive status - Montreal scale for cognitive deficit (Mosa), the level of anxiety and depression – by the HADS scale Results. Patients with TBI consequences of varying severity established significant growth indicators characterizing the level of necrosis and apoptosis of peripheral blood leukocytes, and increase the percentage of cells containing active oxygen species. Reliable dependence of indicators RI + ANV + peripheral blood leukocytes and ROS + on patient's age, duration and type of injury was not found Conclusions. Conditioned upon progression of neurological deficit (accession of extra-pyramidal symptoms, occurrence and progression of cognitive disorders, etc.) significantly higher values RI + ANV + peripheral blood leukocytes and ROS + were found

DISRUPTION OF EXPRESSION OF MRNA HSP27 AND UBIQUITIN AS A MECHANISM OF ESCAPING OF JURKAT LINE TUMOR CELLS FROM APOTOSIS

The research objective is to establish the link between heat shock protein 27 and ubiquitin mRNA expression as well as Jukart tumor cell apoptosis.The method of flow cytofluorometry has been used to evaluate apoptosis realization using FITC-labeled annexin V and propidium iodide along with the amount of reactive oxygen species. Spectrofluorimetry has been applied to register the caspase-3 activity. The content of hydroxyl radicals has been determined by spectrophotometry. The level of ubiquitin and heat shock protein 27 mRNA expression has been identified using real-time PCR. Intact Jukart tumor cells and blood lymphocytes of healthy donors served the material for the research.Following the carried out research it has been found out that the fall in the amount of annexin V positive cells and the reduced caspase-3 activity were accompanied by the rise in the content of hydroxyl radicals and reactive oxygen species against the backdrop of the increased heat shock protein 27 and ubiquitin mRNA expression in Jukart tumor cells.

Immunopathogenetic features of bacterial purulent meningitides

The aim of the study. Comparative assessment of the state of cells of the immune system in patients with purulent bacterial meningitides caused by N.meningitidis and S.pneumoniae . Materials and methods. By the method offlow cytofluorometry there were tested blood and cerebrospinal fluid of 65 patients with bacterial purulent meningitis, with the meningococcal (38 people) and pneumococcal (27 people) nature of the disease. Results. There were revealed general consistencies of the immune response to CNS infection with meningococcus andpneumococcus: the fall of the number of CD16+ CD56+(NK) and the gain in the number of CD19+ cells in the blood, an increase in the absolute number of T lymphocytes, especially, CD3+ CD8+ and NK. In pneumococcal meningitis there was noted the more pronounced upsurge in the absolute number of the NKT (CD3+CD56+) and B-lymphocytes in the blood whereas in cerebrospinal fluid - the more pronounced upturn in the absolute number of NKT that testified to the severe course of the disease. Conclusion. As a result of performed studies there was identified a series of consistencies for the development of the immune response in bacterial purulent meningitides of meningococcal and pneumococcal etiology, which show the high informativeness of the study of immunograms of cerebrospinal fluid in these diseases as compared with blood. In the latter case, immunological studies may contribute not only to the identification of general consistencies of the development of the immune response in bacterial meningitis, but also serve as a basis for the differentiation of the impact of meningococcal and pneumococcal infection on immunocompetent cells, as well as to reflect the severity of the course of the disease, affecting the nature of the therapy.

Cell Cycle Arrest in the Thymus and Spleen in Male Mice under Conditions of Chronic Social Defeat Stress: Effects of Diazepam

The effects of chronic social defeat stress on the percentage of cells in different phases of the cell cycle and in apoptosis in the thymus and spleen of male mice were studied by the method of flow cytofluorometry. In stressed males, thymus weight decreased, the percent of proliferating thymocytes was significantly lower, and the percentage of G0-G1 cells was higher than in intact males. Stress substantially reduced the percentage of splenocytes in the G0-G1 phase and apoptotic cells, but the percentage of S and G2-M cells and proliferation index significantly increased. Chronic administration of anxiolytic diazepam prevented the majority of the changes in the percentage of cells in different phases of the cell cycle, but apoptosis in the thymus increased under these conditions. Possible association between cell cycle disorders, impairment of cell immunity, and chronic anxiety developing under conditions of long-term social defeat stress is considered.

Detection of Inflammatory Processes during Various Diseases by the Method of Flow Cytofluorometry

Oxidative (respiratory) burst is an important manifestation of inflammation. Precise quantitative assessment of this reaction by flow cytometry made it possible to record and evaluate the severity of the inflammatory processes in a wide spectrum of diseases including diphtheria, hepatitis, pneumonia, bronchial asthma, arthritis, vasculitis, postoperative complications, tuberculosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and myocardial infarction. This approach can be employed as a highly sensitive method of detection of inflammatory reactions and monitoring of their course in various pathological processes.

CD24 and CD70 as differential markers of human hepatocellular carcinoma cells in culture

We developed a new method for evaluation of the purity of cell population in primary cultures of hepatocellular carcinoma. Expression of potential surface molecular markers on primary cultures of renal tumors was assayed by the method of flow cytofluorometry. CD24 and CD70 were identified as differential markers, which allowed us to distinguish cancer cells from tumor stromal cells in vitro. A negative marker CD90 was expressed on fibroblasts of the tumor stroma, but not on cancer cells. Combined use of these three markers allows evaluation of the severity of tumor culture contamination with stromal cells.

Assessing the toxic effect of 2,4,6-trinitrotoluene on cells of Escherichia coli K12 by flow cytofluorometry

The magnitude of transmembrane potential delta psi in cells of Escherichia coli K12 was determined by the method of flow cytofluorometry for different phases of growth. It was large in the log phase, whereas in the lag and stationary phases, the population was shown to consist of two subpopulations with low and large values of delta psi in cells. In the presence of 2,4,6-trinitrotoluene (TNT), this bimodal distribution of delta psi over the population was observed during the entire growth period until TNT was almost completely eliminated from the cultivation medium (to a concentration of 18-20 mg/l). The mean value of delta psi in cells of the population grown in the presence of TNT was substantially smaller than that in controls due to the larger size of the subpopulation with a low value of delta psi. Upon elimination of TNT, the distribution of delta psi in cells of the culture became unimodal and close to that in the control culture in the early log phase of growth. These findings are discussed from the standpoint that considers heterogeneity of the culture of Escherichia coli K12 as a mechanism of its adaptation to the presence of xenobiotics.

Cytofluorometric analysis of phenotypes of human bone marrow and umbilical fibroblast-like cells

Comparative analysis of the expression of some surface markers of human bone marrow mesenchymal stem cells, umbilical fibroblast-like cells, and skin fibroblasts was carried out by the flow cytofluorometry method. Mesenchymal stem cells and umbilical fibroblast-like cells were similar by the levels of expression of the main histocompatibility complex antigens, adhesion molecules, and some growth factor receptors. The profile of skin fibroblast surface antigens was characterized by higher expression of the markers typical of differentiated cells. The results prove the possibility of using umbilical fibroblast-like cells as an alternative source of mesenchymal stem cells for cell replacement therapy.

Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope

Apoptosis of placental trophoblast cells has become the subject of intensive research. Recently, a monoclonal antibody (M30) directed against a neo-epitope of cytokeratin 18, that is formed after cleavage of this cytoskeletal protein by caspases, was shown to be of advantage over other tests for the detection of trophoblast cell apoptosis. In the present study, we describe a method for the enrichment of highly pure villous trophoblast cells based on the proteolytic digestion of placental tissue, density gradient separation of dissected cells, and immunoelimination of contaminating, non-trophoblast cells employing an antibody to the HLA class I antigen. The high purity (94-99%) of the trophoblast cell preparation was shown by antibody staining for cytokeratin 7 and absence of vimentin. Furthermore, we demonstrate that after a simple permeabilization and fixation step with 90% methanol and using the M30 CytoDeath, FITC-conjugated antibody, apoptotic trophoblast cells could be distinguished from non-apoptotic cells by flow cytofluorometry in a highly quantitative and sensitive fashion. Our protocol is an improvement over previously used methods such as immunocytochemistry as it allows to differentiate rapidly between competent and apoptotic trophoblast cells by the quantitative method of flow cytofluorometry.

New approach to vital analysis of functional activity in ABC transporters (markers for multidrug resistance) in solid tumors by the method of flow cytofluorometry.

We developed and described a new approach to vital analysis of functional activity of multidrug resistance markers (ABC transporters) in intact biopsy specimens from human solid tumors by the method of flow cytofluorometry. The algorithm of the study underwent revision, and the cell suspension was obtained in the final stage. Intensification of intracellular doxorubicin accumulation (fluorescence) and increase in the number of fluorescent cells and total fluorescence of doxorubicin-accumulating cells produced by ABC transporter inhibitor sodium azide served as the criteria of expression of these transporters in tumor tissue. Informative value of changes in various parameters of doxorubicin fluorescence is discussed. The increase in the count of fluorescent cells in the suspension of tumors cells after treatment with the inhibitor indicates the presence of tumor cells absolutely resistant to this preparation. The proposed method is technically simple, suitable for structurally different tumors, requires small amounts of the biopsy material and, therefore, can be used for routine analysis. The results of our analysis and spectrofluorometric assay of ABC transporters agree very closely, which suggest that this method is adequate for the purpose.

Tumor-specific changes in mouse serum during Ehrlich carcinoma growth.

Using the method of flow cytofluorometry we found that proteinase activity eliminating antigenic determinants from the surface of tumor cells disappeared from the serum of mice with Ehrlich carcinoma. This activity towards Ehrlich carcinoma cells is present in the sera of mice without tumors and in mice with other transplanted tumors. The serum from mice of one strain with Ehrlich carcinoma showed no protease activity against Ehrlich carcinoma cells in mice of other strain. Hence, Ehrlich carcinoma growth is associated with tumor-specific changes in the serum resulting in disappearance of specific protease activity of the serum against tumor cells.

Binding of leukaemic blasts to various subpopulations of lymphokine-activated killer cells.

Conjugate formation by natural killer (NK)-resistant and NK-sensitive leukaemic cell lines with fresh and IL-2-stimulated (lymphokine-activated killer, LAK) peripheral blood lymphocytes (PBL) was studied by a flow cytofluorometry method with double staining. A significant difference in binding of NK-resistant T-cell lymphoma (HuT 78) and NK-sensitive myeloid (K562) blasts to fresh PBL was observed (P < 0.01). Activation of lymphocytes with IL-2 resulted in a significant increase of binding and killing of both K562 and HuT 78. However, in the case of blasts from NK-resistant cell line Daudi a similar conjugate formation with fresh PBL and LAK effectors was observed, despite a significant increase in killing. Various subpopulations of LAK effectors (CD3, CD16 and CD56 positive) displayed similar binding activity towards myeloid (K562) and lymphoid (Raji) blasts, which shows that conjugate formation occurs not only with NK-cell-derived, but also with T-cell-derived LAK cells. The blocking of CD71 antigen (transferrin receptor) on K562 blasts inhibited binding of cytotoxic lymphocytes, which was mostly due to the blocking of binding of CD56+ subpopulation. Our results indicate that the resistance of leukaemic blasts to cell-mediated cytotoxicity may depend on different (and probably several) steps of this process.

Kinetic analysis of cytotoxic lymphocyte—target cell interaction as quantified by dual parameter flow cytometry

The kinetics of conjugate formation between leukemic cell lines (K562 and Daudi) and lymphokine-activated killer (LAK) cells was studied. A flow cytofluorometry method using double immunofluorescence staining was applied. During the first 15 min of incubation of LAK effectors with leukemic targets, a rapid binding occurred, followed by a plateau phase lasting until 30 min of observation. A considerable, yet not statistically significant, between-donor variability was noticed. A mathematical model of conjugate formation kinetics, based on the analogy to enzyme kinetics, was formulated and validated. Parameters of the model were related to the binding capacity of effector and target cells, and to the lifetime of conjugates and free cells. The concordance of theoretical curves with experimental data proved that the described model can be considered as a useful tool for the evaluation of kinetic and dynamic characterization of conjugate formation between leukemic targets and LAK effectors.