scholarly journals Features of immunophenotypic finding B-cell lymphoproliferative diseases by flow cytometry

2020 ◽  
Vol 101 (1) ◽  
pp. 145-152
Author(s):  
J J Chuksina ◽  
E V Kataeva ◽  
T A Mitina
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Information Content ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Differential Diagnostics ◽  
Lymphoproliferative Diseases ◽  
Color Flow ◽  
Positive Expression ◽  
Hodgkin's Lymphomas

Aim. To assess the information content of conventional and additional immunophenotypic markers (CD200, CD305) in the differential diagnosis B-cell lymphoproliferative diseases by flow cytometry. Methods. An immunophenotypic study using 4-color flow cytometry was performed in 204 patients with different variants of B-cell non-Hodgkin's lymphomas. The study material included peripheral blood and bone marrow. The expression of CD45, CD19, CD20, CD22, CD79b, CD79a, CD5, CD10, CD23, FMC7, CD43, CD38, CD11c, CD103, CD25, CD 200, CD 305, light chains of immunoglobulins (kappa/lambda) using monoclonal antibodies (Becton Dickinson, USA) was evaluated. The intensity of antigen expression was assessed using mean fluorescence intensity (y. e.). Results. Conventional FMC7-positive expression revealed only half patients with different variants of leukemization of non-Hodgkin's lymphomas, whereas atypical positive expression of CD23 was observed in patients with marginal spleen lymphoma and follicular lymphoma in 27.3 and 28.6% of cases, respectively. In mantle cell lymphoma, expression of CD200 in B-cell was detected in a significantly smaller number of observations, accompanied by a significant decrease in the average intensity of CD200 fluorescence compared to B-cell chronic lymphocytic leukemia (B-CLL) cells. The mean fluorescence intensity (MFI) of CD305 in hairy cell leukemia is significantly higher than in splenic marginal zone lymphoma (SMZL) with villous lymphocytes. Conclusion. Different levels of the information content of some conventional markers were revealed in differential immunophenotypic diagnosis of B-cell lymphoproliferative diseases by flow cytometry; the use of additional markers CD200 and CD305 was highly informative in differential diagnostics between different variants of B-cell lymphoproliferative diseases with similar immunophenotypic and morphological characteristics of lymphoid elements.

scholarly journals Comparative analysis of surface membrane immunoglobulin determination by flow cytometry and fluorescence microscopy.

1986 ◽  
Vol 34 (4) ◽  
pp. 475-481 ◽  
Author(s):  
D F Gebhard ◽  
A Mittelman ◽  
C Cirrincione ◽  
H T Thaler ◽  
B Koziner
Keyword(s):  
Flow Cytometry ◽  
Standard Deviation ◽  
Fluorescence Microscopy ◽  
B Cell ◽  
Membrane Surface ◽  
Surface Membrane ◽  
Flow Cytometric Analysis ◽  
Normal Individuals ◽  
The Mean ◽  
Hodgkin's Lymphomas

The analysis of membrane surface immunoglobulin (SmIg) on B lymphocytes was carried out in 59 normal individuals and nine patients with B-cell non-Hodgkin's lymphomas by conventional immunofluorescence microscopy and flow cytometry. Five channel settings of a cytofluorograph were evaluated (100, 150, 200, 250, 300) and the mean and standard deviation of the percent positive cells were calculated and compared to the mean and standard deviation of the microscope reading. On the basis of the relative fluorescence reactivity, we were able to determine a fluorescence intensity at which the results of flow cytometry and fluorescence microscopy were comparable. In normal individuals, for cells expressing surface Ia, the channel giving similar results to that of fluorescence microscopy was 150; for kappa and lambda chains, channel 200; for Fab'PV, channel 200; and for IgM, channel 250. In patients with B-cell non-Hodgkin's lymphomas, for cells expressing surface Ia the channel giving similar results to that of fluorescence microscopy was 100; for kappa, channel 100; for lambda, channel 200; for Fab'FV, channel 150; and for IgM, channel 150. Flow cytometric analysis of SmIg appears to be superior to fluorescence microscopy in efficiency, and has the added advantages of being a rapid, sensitive, and objectively quantitative methodology.

The BLyS Survival Pathway in NHL-B Cells: Constitutive NF-kB and NFAT Lead to Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2279-2279
Author(s):  
Lingchen Fu ◽  
Tamayo Archito ◽  
Yen-Chiu Lin-Lee ◽  
Lan Pham ◽  
Linda Yoshimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Low Grade ◽  
Rt Pcr ◽  
Protein Levels ◽  
Realtime Pcr ◽  
Survival Pathway ◽  
Hodgkin's Lymphomas

Abstract B-cell non-Hodgkin’s Lymphomas (NHL-B), neoplasms of the immune system have shown a significant increase in incidence in the USA over the last three decades. While the pathophysiology of the NHL-B is still unclear, the need to identify the relevant genes and critical signaling pathways, and their involvement in the disease processes in NHL-B have begun to be elucidated. Recently, B Lymphocyte Stimulator (BLyS) has been described as a relatively new member of the TNF ligand family, as a potent cell survival factor that is expressed in many hematopoietic cells, including neoplastic B cells. BLyS can bind to three receptors: TACI, BCMA, BAFF-R, and plays a critical role in B cell maturation, differentiation and proliferation. Relatively high levels of BLyS has been found in the serum of NHL-B patients as well as of the patients with autoimmune disease. The mechanisms of BLyS gene expression and regulation is still unclear, but we have recently found that BLyS is constitutively expressed in several NHL-B cell lines and patient tumor samples by RT-PCR, confocal microscopy, realtime PCR and flow cytometry (FCM). We detected high levels and differential expression of BLyS receptors (TACI, BCMA, BAFF-R) in several NHL-B cell lines by flow cytometry, RT-PCR and realtime PCR in both NHL-B cell lines and patient tumor samples. We have identified a single binding site for NF-kB and two binding sites for NFAT in the BLyS promoter. We also show in aggressive lymphoma B cells that constitutive NF-kB and NFAT binds to the BLyS promoter constitutively. Inhibiting NF-kB/NFAT activity levels, using the NF-kB inhibitors, BAY-11 or Velcade (PS-341), can decrease NF-kB binding activity in the BLyS promotor by EMSA. These inhibitors also decrease BLyS and BAFF-R mRNA and protein levels by realtime PCR and flow cytometry. Similarly, when NHL-B cells were transfected with dominant negative NFAT or NF-kB constructs, there is a 50% decrease in BLyS and BAFF-R expressions, demonstrating that both the ligand (BLyS) and the receptor (BAFF-R) expression are regulated by NFAT and NF-kB. Interestingly, follicular (low grade) lymphoma cells do not express constitutive NF-kB/NFAT activation, and barely detectable mRNA and protein levels of BlyS, but can be activated with exogenous CD154/anti IgM in vitro, activating NF-kB/NFAT and promoting binding to the BLyS promoter by EMSA. This results in a significant increase of BLyS protein level by flow cytometry. Our studies indicate that constitutive NF-kB and NFAT are critical transcriptional regulators of the BLyS survival pathway in malignant B cells that may provide a future therapeutic target in the aggressive NHL-B.

Standardization of Functional Immune Cell-Based Assays: An Integral Aspect to Vaccine and Biologic Development and Validation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3933-3933
Author(s):  
Julie Wilkinson ◽  
Cecilia Smith ◽  
Sybil D’Costa ◽  
Enrique Rabellino
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Sample Preparation ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Immune Cell ◽  
Cell Populations ◽  
Surrogate Markers ◽  
Functional Assays ◽  
Variable Nature

Abstract The utility of the ex-vivo evaluation of immune cell functionality in the context of a) Determining an efficacious vaccine strategy for infectious diseases/cancer, b) Determining a tolerance profile in autoimmunity and transplantation, and c) understanding the basic mechanisms of immune cell responses in disease pathogenesis is well recognized. However, the benefit of these assays as surrogate markers of immune cell activity in vivo has not been fully realized due to the variable nature of these in vitro assays which is particularly pronounced in T cell functional assays. This variability arises from a variety of factors ranging from choice of assay, source of the cells, the sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol for the chosen assay and ultimately data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated. A 5-color flow cytometry assay (2–3 surface markers; 2 intracellular markers) was developed to characterize the restricted polyclonal (SEB/CD28) and antigen specific (CEF peptide pool) cytokine and cytotoxic profile response in human PBMCs. A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of stimulated human peripheral blood mononuclear cell populations (PBMC) for flow cytometric analysis. Statistically significant reductions in both inter and intra assay variability was observed in the automated methodology as compared to the manual assay with improvements in CVs for positive cell numbers and mean fluorescence intensity. For example, the inter assay CVs for IFNg cytokine producing CD4+ T cell populations improved from approximately 15 to 5, while the mean fluorescence intensity improved nearly 5 fold with automation. Importantly, the automated methodology furnished comparable responses in percent positive cytokine/cytotoxicity profiles as compared to the manual method while reducing the “handson” sample preparation and analysis time from 2 hours to 20 minutes. With the standardization of functional assays, other sources of variability in assays result can now be addressed specifically e.g. specimen handling, freezing, thawing, culturing, or biological. Standardized multiparametric functional profiling of the cells thus reveals the complex nature of the immune response and lends credence to their use as surrogate markers of efficacy and functionality.

The Prevalence of Chronic Lymphoproliferative Diseases in Patients with Borderline Lymphocytosis in Community Practice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4975-4975
Author(s):  
Zuhair Y. Ghanem ◽  
Mahmoud Q. Moammar ◽  
Sherif A. Nasr ◽  
Maher Albitar
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Early Diagnosis ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Lymphoproliferative Disease ◽  
Lymphocytic Leukemia ◽  
Low Grade ◽  
Community Practice ◽  
Lymphoproliferative Diseases

Abstract Introduction: The standard criterion for diagnosing chronic lymphocytic leukemia (CLL) is clonal lymphocytosis of >5×10 9/L. Cases of CLL with normal lymphocyte count have been diagnosed by flow cytometry based on the presence of clonal CD19+/CD5+/CD23+ cells. Therefore, it is not unexpected that a proportion of patients with borderline lymphocytosis (>3.5 and <5.0x10 9/L) will have CLL. The aim of our study is to establish the prevalence of CLL in patients with borderline “4.0 to 5.0x109/L” lymphocytosis in the adult population (age >40 years) seen in community practice. Methods: Using flow cytometry we analyzed a total of 157 sequential peripheral blood samples collected from patients older than 40 years presented with borderline lymphocytosis (4 to 5 x109/L). Majority of these patients (#106) were detected incidentally during routine CBC and 51 samples were submitted to rule out lymphoproliferative diseases. Results: Forty of the 157 (26%) patients had clonal B-cell disease meeting the criteria for chronic lymphoproliferative disease. The disease was classified as CLL in 35 patients (87.5%), hairy cell leukemia in 1 patient (2.5%), Waldenstrom’s macroglobulinemia in 1 patient (2.5%) and marginal zone B-cell lymphoma in 3 patients (7.5%). This data suggests that patients older than 40 year with lymphocyosis >4x10 9/L have high probability of having chronic lymphoproliferative disease. This disease could be other than CLL and should be thoroughly investigated. DISCUSSION: In our study the prevalence of low grade lymphoproliferative disorders in patients with borderline lymphocytosis (4–5 x109/L) above the age of 40 is 26%. This number may be positively skewed considering our selection criteria (including a subset of patients retrospectively included). Currently, there is no data to support that early intervention is beneficial for CLL, even for patients with unfavorable prognosis (e.g., those with ATM and P53 deletions). Early diagnosis of CLL will create more opportunity to study the disease in its early stages.[Shanafelt TD, Geyer SM, Kay NE: Prognosis at diagnosis: integrating molecular biologic insights into clinical practice for patients with CLL. Blood. 2004 Feb 15;103(4):1202–10. Epub 2003 Oct 23. Review.], [ Hamblin TJ: Achieving optimal outcomes in chronic lymphocytic leukemia. Drugs. 2001;61(5):593–611. Review.]. Since lymphocyte doubling time (LDT) is a prognostic factor (the prognostic utility of LDT is most important for patients with early stage disease who typically are treated by watchful waiting [ Shanafelt TD, Call TG. Current approach to diagnosis and management of chronic lymphocytic leukemia. Mayo Clin Proc. 2004 Mar;79(3):388–98. Review. ] ). early diagnosis may help to better segregate low from high-risk patients. Finally, in today’s cost containment pressures, it is beneficial to have an expectation for the cost/benefit ratio of performing a test. Knowing the prevalence of disease at decision limits may help us to better justify establishing testing guidelines.

Evidence of Pre-Diagnostic Monoclonality in Blood Obtained up to 6.4 Years Preceding Diagnosis of Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3082-3082
Author(s):  
Ola Landgren ◽  
Maher X. Albitar ◽  
Wanlong X. Ma ◽  
Fatima R. Abbasi ◽  
Richard B. Hayes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Gene Family ◽  
B Cell ◽  
Suppressor Gene ◽  
Blood Draw ◽  
Color Flow ◽  
Mutation Status ◽  
Diagnostic Samples ◽  
Vh Gene ◽  
Clinical Records

Abstract Background: Monoclonal B-cell lymphocytosis (MBL) is reported in about 3% of the general adult population 65 yrs or older. Risk of progression from MBL to CLL requiring treatment has been estimated to about 1% per yr. For the first time we established the occurrence of preceding MBL using prospectively collected pre-diagnostic samples from CLL pts. Also, we tested the hypothesis that indolent (IgVH mutated) CLL occurs after MBL while aggressive (IgVH unmutated) CLL has an alternative route that develops rapidly or bypasses MBL. Methods: Using the population-based nationwide PLCO (Prostate, Lung, Colorectal and Ovarian) cancer screening trial (n>144,500 cancer-free adults), we identified 46 CLL pts with available baseline cryopreserved whole blood obtained 3 to 77 months prior to CLL diagnosis. Clinical information was retrieved from screening questionnaires and medical records. 10 healthy adult controls were included. 6-color flow cytometry (CD45/CD19/CD5/CD10/kappa/lambda) and reverse transcription/polymerase chain reaction (RT/PCR) for IgH gene were used to determine evidence of expression of monoclonal IgH gene family and to establish the VH gene mutation status. Direct sequencing without subcloning (for clonal cases) and with subcloning (for non-clonal cases) was used to determine mutation status. We sequenced exons 5, 6, 7, 8, and 9 of the p53 suppressor gene for mutations. Results: After exclusion of one subject with absent viable cells, we analyzed pre-diagnostic blood specimen from 45 CLL pts. CLL diagnosis was based on clinical criteria and confirmed by review of clinical records. Immunophenotyping data were available for 39 (87%) pts. The Rai CLL stage distribution was: 0 (n=26), 1 (n=9), 2 (n=2), 3 (n=1), missing/unclear (n=7). The mean time between pre-diagnostic blood draw and subsequent CLL diagnoses was 3 yrs. Using flow cytometry, sequencing, and PCR, we demonstrated evidence of monoclonality in 43/45 (96%) pre-diagnostic samples from CLL pts. Among individuals whose blood was obtained <3 yrs vs. >3 yrs prior CLL diagnosis, the proportion of pre-diagnostic monoclonality was 28/28 (100%) vs. 15/17 (88%), respectively; the proportion of IgVH mutated pre-diagnostic clones was the same in both groups: 23/28 (82%) vs. 14/17 (82%). The most frequently expressed VH gene family was VH3 (40%) followed by VH4 (29%), VH1 (9%), VH5 (9%), VH2 (7%), and VH6 (2%), with no expression of VH7 gene families. We found 2 pts with mutations in the p53 suppressor gene; one in exon7/M237K, and one in exon 6, P22 that is silent and has been reported as in some tumors and a polymorphism. One sample with unmutated IgVH appeared to have a deletion at p53 in both alleles. Immunophenotyping analyses for pre-diagnostic clones showed kappa n=27, lambda n=8, n=4 biclonal, and missing/indeterminate n=6 (kappa:lambda ratio >3:1). Comparisons of pre-diagnostic and diagnostic kappa/lambda status in the same persons showed 100% concordance. All negative controls lacked evidence of monoclonality. Conclusions: In this first prospective study examining pre-diagnostic blood obtained 3 to 77 months prior to CLL diagnosis, we found a monoclonal B-cell population in virtually all the persons; 82% were IgVH mutated. Our findings suggest that CLL is commonly preceded by the precursor condition MBL.

Strong Cell Surface Expression of the Toll-Like Receptor Homolog CD180 Identifies Circulating Cells of Marginal Zone Lymphoma From Other B-Cell Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1542-1542
Author(s):  
Laurent Miguet ◽  
Lucile Baseggio ◽  
Sarah Lennon ◽  
Pascale Felman ◽  
Luc Fornecker ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Marginal Zone ◽  
Extreme Values ◽  
Marginal Zone Lymphoma ◽  
Toll Like Receptor ◽  
Box Plot

Abstract Abstract 1542 Despite typical features described for marginal zone lymphoma (MZL), the distinction of MZL from other small mature B-cell leukemias/lymphomas (in particular atypical chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) or lymphoplasmacytic lymphoma (LPL) may be still difficult due to the lack of immunological positive markers of MZL and before complementary analysis (histology, karyotype). In order to find new markers, we conducted proteomic analyses on plasma membrane microparticles derived from malignant cells and identified the Toll-like receptor homolog CD180 protein as a candidate marker of MZL. This protein, also called RP105, is a leucin-rich repeat type 1 membrane protein with high extra-cellular homology to the LPS receptor. The relevance of this marker was evaluated in peripheral blood B-cells from 25 normal controls and 91 patients (MZL, n=30; CLL, n=30; MCL, n=16 and LPL, n=15) by flow cytometry. As already described by Porakishvili et al, normal B-cells displayed higher CD180 expression than CLL B-cells (MFI=5940+/− 1708 and MFI=150 +/− 794.4, respectively p<0.001, Mann-Whitney test). We observed also a low CD180 expression in MCL (1292 +/− 1084, p<0.001) and LPL (1443+/− 905.5, p<0.001), not different from CLL and significantly lower than control B-cells (see figure). On the contrary, staining of MZL was not different from controls (7856 +/− 1030, p>0.05), but significantly higher than CLL, MCL and LPL. In addition, among the lymphomas derived from MZ B-cell, CD180 expression was statistically higher in splenic diffuse red pulp lymphoma (SDRPL) than MZL. Intra-cellular staining of CD180 showed a significant positivity in tumoral B-cell tested (CLL, MCL, MZL), suggesting that the protein is produced but its expression at the cell surface is impaired. The reasons of this alteration are not known. Furthermore, cross-linking of this receptor induced a stronger increase of CD86 expression at the cell surface in MZL than in control B-cells and in CLL B-cells, suggesting an activated NF-kB pathway. These results strongly suggest that CD180 may be the first positive immunological marker for MZL, able to distinguish MZL from CLL, MCL, LPL in blood samples. Large prospective studies are needed to precise its diagnosis impact and its expression in the different subtypes of MZL. Figure: CD180 expression of normal and malignant B-cells. The box plot represents the median of mean fluorescence intensity (MFI), 25/75 percentiles and extreme values. Figure:. CD180 expression of normal and malignant B-cells. The box plot represents the median of mean fluorescence intensity (MFI), 25/75 percentiles and extreme values. Disclosures: No relevant conflicts of interest to declare.

IL-10 Contributes to the Development of Immunosuppressive CD14+HLA-DRlow/- monocytes in B-Cell Non-Hodgkin’s Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2979-2979
Author(s):  
Bing Xiu ◽  
Yi Lin ◽  
Deanna Grote ◽  
Steven Ziesmer ◽  
Michael Gustafson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Poor Prognosis ◽  
Underlying Mechanism ◽  
Monocyte Count ◽  
Newly Diagnosed ◽  
Color Flow ◽  
Healthy Donors

Abstract Background - The phenotype and biological role of monocytes in B-cell non-Hodgkin lymphoma (NHL) is not fully understood, however, an increased absolute monocyte count in the peripheral blood of lymphoma patients is associated with a poor prognosis. We have previously reported that monocytes from patients with relapsed B-cell NHL displayed an immunosuppressive CD14+HLA-DRlow/- phenotype that correlated with a poor prognosis. However, the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop in patients with B-cell NHL is unknown. The goal of this study was to determine whether cytokines are responsible for the increased number and phenotype of CD14+HLA-DRlow/-monocytes present in lymphoma patients. Methods – Whole blood from patients with newly diagnosed, untreated B-cell NHL (n=20) and healthy donors (n=20) was stained with a panel of antibodies and analyzed by 10-color flow cytometry. Cytokine levels in serum and culture supernatants were measured by Luminex and ELISA, respectively. Polarization of monocytes from healthy donors was performed by incubating CD14+ cells with specific cytokines or supernatants from B-cell NHL cell lines for 24 hours. Activation and proliferation of CD4+T cells cocultured with IL-10-pretreated monocytes were measured by flow cytometry. Results – By flow cytometry, we observed that the absolute number of peripheral monocytes was increased in newly-diagnosed lymphoma patients compared to healthy donors. A significant proportion of these monocytes displayed an immunosuppressive CD14+HLA-DRlow/- phenotype and the numbers of CD14+HLA-DRlow/- cells were significantly higher in lymphoma patients than in healthy donors. To identify the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop thereby leading to increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL, we tested a panel of cytokines and found that IL-10 played a key role in the process. Firstly, treatment of monocytes with IL-10 in vitro resulted in the generation of CD14+HLA-DRlow/- cells. Second, monocytes co-cultured with IL-10 producing lymphoma B-cells, or treated with supernatants from lymphoma cell cultures, developed a CD14+HLA-DRlow/- phenotype. Thirdly, IL-10 levels were increased in the serum of DLBCL (p<0.0001) and FL patients (p=0.010) compared to healthy controls, and serum IL-10 levels correlated with increased numbers of peripheral monocytes (p=0.02). Finally, IL-10-pretreated CD14+HLA-DRlow/- monocytes were significantly immunosuppressive and inhibited the proliferation and activation of CD4+T cells in co-culture assays. Conclusions- Taken together, our results suggest that IL-10 signaling contributes to the increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL. Strategies to inhibit IL-10 production may therefore have therapeutic potential in B-cell NHL. Disclosures No relevant conflicts of interest to declare.

scholarly journals The High PMNs Phagocytosis Resistance of Enterococcal Isolates from RTx Patients

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Tomasz Jarzembowski ◽  
Agnieszka Daca ◽  
Jacek M. Witkowski ◽  
Ewa Bryl ◽  
Bolesław Rutkowski
Keyword(s):  
Flow Cytometry ◽  
High Risk ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Culture Media ◽  
Immunocompromised Patients ◽  
Bacterial Cells ◽  
Opportunistic Pathogens ◽  
Fluorescent Reporter ◽  
Significant Difference

Infections caused by opportunistic pathogens such as enterococci remain difficult to manage, especially in immunocompromised patients. Because of infections’ limited symptoms in such patients the additional problems are to find proper diagnostic criteria and the management of infection. Here we aimed to compare the resistance of commensal enterococcal strains and RTx patients’ isolates, to PMNs phagocytosis. Thirty-six enterococcal urine and faecal isolates from RTx patients and 17 faecal isolates from healthy volunteers were cultured in planktonic and biofilm forms in 37°C or 42°C. Another tested variable was the addition of immunosuppressant to the culture media. Bacterial cells were stained with fluorescent reporter (CFDA, PI) and incubated with PMNs. Results of phagocytosis were estimated as a mean fluorescence intensity (MFI) of PMNs using flow cytometry. Commensal enterococci cultured in all abovementioned (37°C and 42°C/the addition of immunosuppressant) conditions were less resistant to phagocytosis compared to RTx isolates. Observed significant difference in phagocytosis resistance suggests that patients in immunosuppression are colonized with high risk strains which may lead to the development of infection.

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