[Flow cytometry in microbiology]. / Prutoková cytometrie v mikrobiologii.

Flow cytometry is a method that allows simultaneous measurement and analysis of physical and chemical characteristics of cells or other biological particles during their passage through the laser beam. Although this method is mainly used in the study of cell differentiation and functional analysis of eukaryotic cells, the basic principles of flow cytometry can be applied to microorganisms. Methods based on the analysis of a single cell, such as flow cytometry, in combination with measurement of cell viability using special fluorescent probes allow a deeper insight into the diversity of populations and functioning of microbial communities and also facilitate understanding the phy-siological diversity of seemingly similar acting populations. When using specific fluorescent dyes for the selective labeling of selected species of microorganisms, the method is potentially very specific. The aim of this paper is a brief overview of applications of flow cytometry, which can be used in microbiology.

Quantiferon-CMV test in prediction of cytomegalovirus infection after kidney transplantation.

Infection with cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunosuppressed patients, including organ and bone marrow transplant recipients. The majority of CMV disease is caused by reactivation of alatent infection rather that by newly acquired virus. Many techniques have been currently available to aid in the diagnostics of CMV disease. In this report we performed a prospective evaluation of Quantiferon-CMV assay (Cellestis) to determine whether the test is predictive of CMV disease. CD8+ T-cell CMV-specific immunity was assessed in a longitudinal cohort of 14 kidney transplant recipients. According to our data, subjects with higher cellular immune response measured with Quantiferon test had a lower risk of manifestation of CMV infection than subjects with lower responses. Despite the small number of patients and large intra- and interindividual variability of the data in the study, we observed the Quantiferon-CMV assay to be a sensitive specific test to detect a virus-specific T-cell response. We propose that this assay in combination with viral DNA load estimates may prove to be useful to stratify patients at risk of CMV disease.

Immune system parameters in children of Central and Eastern Europe: the CESAR study.

OBJECTIVE: of this paper is to compare observed values of immune parameters obtained in the CESAR study (The Central Europe Study of Air Pollution and Respiratory Health, funded by EC PHARE program) with ranges derived from other large population-based studies. STUDY DESIGN: Data were collected in healthy school children aged 9-11 years, in 6 countries: Bulgaria, the Czech Republic, Hungary, Poland, Romania and the Slovak Republic with the same standard approach in 1996. Random samples of 85 children per country, from 19 communities were selected from children having completed the health questionnaire, in total 495 children were analyzed. Lymphocyte subsets were determined by two-colour flow cytometric immunophenotyping using the lysed whole blood method (Becton-Dickinson). For determination of immunoglobulin concentration in sera nephelometric method (Behring Nephelometer system) was used. RESULTS: Medians, (5th-95th percentiles) of the lymphocyte subsets absolute count (x 10(9)/l) were as follows: CD19+ B cells 0.36 (0.13-0.66), total CD3+ T cells 1.74 (0.98-2.90), CD3+CD4+ helper-inducer T cells 0.95 (0.47-1.78), CD3+CD8+ suppressor/cytotoxic T cells 0.71 (0.38-1.22), CD3-CD16+56+ NK cells 0.36 (0.14-0.78), and for CD3+CD4+/CD3+CD8+ ratio 1.4 (0.8-2.4). Medians, (5th-95th percentiles) of percentages of lymphocyte subpopulations (%) were as follows: CD19+ B 13 (7-22), CD3+ T 70 (59-80), CD3+CD4+ T 38 (27-48), CD3+CD8+ T 28 (20-39), CD3-CD16+56+ NK cells 14 (6-27). Medians, (2.5th-97.5th percentiles) of the total immunoglobulin [g/l] were 11.7 (7.4-18.2) for IgG, 1.2 (0.5-2.5) for IgM, and 1.5 (0.5-3.4) for IgA. Based on the aspects of the size of the CESAR immune biomarker study and on the use of the standardized protocols we recommend to use the reference ranges on lymphocyte subsets and immunoglobulin in Europe as provided by this study.

Immune biomarkers in relation to exposure to particulate matter: a cross-sectional survey in 17 cities of Central Europe.

Human population data on air pollution and its effects on the immune system are scarce. A survey was conducted within the framework of the Central European Study of Air Quality and Respiratory Health (CESAR) to measure a panel of immune biomarkers in children of Bulgaria, Czech Republic, Hungary, Poland, Romania, and Slovakia. Seventeen cities were chosen to represent a wide range of exposure to outdoor air pollution. In each, ambient particulate matter of less than 10 microns diameter and less than 2.5 microns diameter (PM10 and PM2.5) were measured with a Harvard impactor. Blood was collected from 366 school children aged 9 to 11 yr between 11 April and 10 May 1996. The percentage of B, total T, CD4+, CD8+, and natural killer (NK) lymphocytes was determined by flow cytometry (Becton Dickinson); total immunoglobulins of class G, M, A and E (IgG, IgM, IgA, and IgE) were measured in serum using nephelometry (Behring). Associations between PM and each log-transformed biomarker concentration were studied by linear regression, in a two-stage model. The yearly average concentrations varied from 41 to 96 micrograms/m3 for PM10 across the 17 study areas, from 29 to 67 micrograms/m3 for PM2.5, and from 12 to 38 micrograms/m3 for PM10-2.5 (coarse). Number of B, CD4+, CD8+, and NK lymphocytes increased with increasing concentration of PM, having adjusted for age, gender, parental smoking, laboratory of analysis, and recent respiratory illness. Differences in lymphocyte number were larger and statistically significant for exposure to PM2.5. Similar results were found when we examined the association between PM and lymphocyte number separately for each laboratory. Total IgG was increased with increasing concentration of PM, significantly in the case of PM2.5. When we repeated the analyses with two other statistical approaches the results did not differ from those reported here. The effect of coarse PM on lymphocyte numbers appears small in comparison to PM2.5. One possible interpretation of our findings is that long-term exposure to airborne particulates leads to inflammation of the airways and activation of the cellular and humoral immune system.

Evans' syndrome in a child with diabetes mellitus.

A patient suffering from infantile-onset insulin-dependent diabetes mellitus is reported in whom immune pancytopenia (Evans' syndrome) developed at the age of 2 1/2 years. Hepatosplenomegaly, chronic lymphadenopathy, and elevated levels of immunoglobulins G and M were also present. The course of Evans' syndrome was fatal in this patient. The association of Evans' syndrome with other immune disorders is discussed.

Antiimmunoglobulins of rheumatoid factor (RF) type in prediction of melanoma patients.

It was found out the titer of antiimmunoglobulins of the rheumatoid factor (RF) type in the sera of melanoma patients can be used in prediction of the development of the patients' status. High titers of antiimmunoglobulins of the RF type, determined by the latex-fixation test (LFT), indicate an unfavorable prognosis in patients with clinical manifestation of uncontrollable recurrence appearing from 1.5 to 2 years after occurrence of the LFT titer.