Constitutive activation and accelerated maturation of peripheral blood T cells in healthy adults in Burkina Faso compared to Germany: the case of malaria?

OBJECTIVE: It is not exactly known how frequent exposure to Plasmodium falciparum shapes the peripheral blood T-cell population in healthy West Africans. METHODS: The frequency of peripheral blood CD4(+) lymphocytes responding to Plasmodium falciparum merozoite surface protein 1 (PfMSP-1) by production of interferon-gamma (IFN-γ), interleukin-2 (IL-2) or tumor necrosis factor-alpha (TNF-α) was determined using a commercially available flow cytometric activation assay (FastImmune) in 17 healthy adults in Nouna, Burkina Faso. T-cell activation and maturation in peripheral blood of healthy adults in Burkina Faso (n=40) and Germany (n=20) were compared using immunophenotyping and three-colour flow cytometry. RESULTS: Significant numbers of PfMSP-1 -specific CD4(+) lymphocytes producing IFN-γ, IL-2 and/or TNF-α were detected in 14 healthy adults in Nouna. Cytokine profiles showed predominant production of IFN-γ and TNF-α. Compared to Germans, Burkinabé showed markedly lower proportions of CCR7+ CD45RA+ naive CD4(+) cells and slightly higher frequencies of CD95(+)CD4(+) T-cells and of CD38(+) CD8(+) T-cells. The median antibody-binding capacity of CD95(dim) CD4(+) T-cells in Burkinabé was more than twice the value observed in Germans (263 vs. 108 binding sites per cell, p<0.0001). CONCLUSIONS: We hypothesize that an IFN-γ-induced increase in the expression level of CD95 on CD4(+) lymphocytes may lower the activation threshold of resting naive CD4(+) T-cells in healthy adults living in Burkina Faso. Bystander activation of these cells deserves further study as a molecular mechanism linking strong IFN-γ responses against Plasmodium falciparum to decreased susceptibility to parasitemia observed in specific ethnic groups in West Africa.

Multiparameter absorption measurements in automated microscopy. Simultaneous quantitative determination of DNA and nuclear antigen.

OBJECTIVE: To test a dual DNA-nuclear antigen staining method for multiparameter absorption image analysis. STUDY DESIGN: MCF 7 cells, grown on glass slides, served as a model to test the staining technique. For DNA, Feulgen-based CAS quantitative DNA staining, and for nuclear antigen, alkaline phosphatase-based immunocytochemical staining with CAS Red as the chromogen, were used. MIB-1, estrogen and progesterone receptors were used as examples of nuclear antigen staining. Measurements were performed with the DISCOVERY image analyzer. RESULTS: Scatterplots, in which the nuclear antigen content was plotted against the DNA content, were obtained. Immunostain-positive and -negative populations could be discriminated. These cells were visualized in image galleries. The DNA histograms of the positive and negative cells showed no change in coefficient of variation or integrated optical density ratio of the G0, G1 and G2 + M peaks as compared to single DNA staining. The intensity of the immunostain increased as compared to the single immunostaining result. CONCLUSION: This staining technique allows the simultaneous accurate measurement of costained DNA and antigen within the same nucleus. This opens the possibility for studies in which nuclear antigen expression is monitored during the cell cycle or in cells of different ploidy classes. Identified cells can also be visualized by presentation in an image gallery or by relocation on the slide. This can support the analysis of clinical samples, where cytometric data can be correlated with and confirmed by visual diagnosis.

CytosafePLUS. A workstation for screening, supervision, reviewing, quality assurance and education in cytopathology.

In pathology, the subjective cytologic and histologic diagnosis suffers from large observer variability. Effective methods of reducing the effects of this observer variability in cytologic diagnosis are close supervision of the diagnostic process and multiple screening by different observers of cases that have a higher risk of abnormalities as well as closely supervised follow-up procedures for early lesions. Accurate supervision of screening completeness has become possible with the development of computer programs and microscope instrumentation to register the path of the objective over the slide and therefore the fields that have been inspected by the screener (Axio-Home, Carl Zeiss, Oberkochen, Germany; Navigator, Becton Dickinson Cellular Imaging Systems, Leiden, the Netherlands). Based on these technologies, all objects of interest on the microscope slide are marked electronically, and the X,Y coordinates of these marks are stored in the computer. With these coordinates, a motor-driven microscope stage can be redirected to the selected objects, enabling the observer to quickly review the objects. The reviewer can thus reinspect cells or groups of cells, previously identified, in a much more accurate way than is presently possible, by marking objects of interest with ink dots. To these marked objects, specific diagnoses can be attached, as can comments or questions, which are also stored in the computer and are made part of the cytology report. After analysis, all marked objects can be relocated automatically with the help of the computer-directed microscope stage. Using this concept, a subsequent observer or a supervisor can easily and effectively reply to the remarks attached to these objects. It is also possible to reedit the comments made or to attach answers to specific questions. In an interobserver study, encompassing 50 cases of columnar cell abnormalities screened by five observers using the Navigator microscope stage and software, it proved possible to reduce reviewing time significantly. On the basis of marked cytologic characteristics discriminating cellular features in the diagnosis, endocervical columnar abnormalities could be identified. The coordinates of the objects of interest are made part of the laboratory record of the cytology findings (Cytosafe, Omnisys, Hoevelaken, the Netherlands). This development largely facilitates the review of previous findings when evaluating follow-up specimens. With the use of a Navigator motor-driven stage and CCD camera, microscope images can be made of the specimen under study. These images can be stored in the computer in a digitized format, labeled with their X,Y coordinates on the specimen. The CytosafePLUS workstation, encompassing the Navigator automated cell positioning system and CCD attachment, is a highly effective tool in cytologic screening, reviewing and reporting, in surveillance of follow-up, in intralaboratory and interlaboratory consultation, in cytology training, in proficiency testing and in quality assurance.

Soluble CD23 measurement by CBA: a convenient and reliable quantification method in chronic lymphocytic leukemia.

The soluble form of the transmembrane glycoprotein CD23 corresponding to the low-affinity receptor for the immunoglobulin E (sCD23) is found in the serum of patients with chronic lymphocytic leukemia (CLL). In this disease, an increase in sCD23 level is predictive of poor prognosis at diagnosis as well as during clinical outcome. Quantification of sCD23 is classically performed by enzyme-linked immunosorbent assay (ELISA), a method not routinely used in hematology laboratories. Our aim was to apply cytometric bead array (CBA) technology to measure sCD23 levels. We tested 420 serum samples, 360 from patients and 60 from healthy volunteers. We selected three pairs of monoclonal antibodies recognizing the CD23 molecule that were tested in various conditions of temperature, centrifugation, washing, or chemical supplementation. Satisfactory performances in terms of repeatability (CV: 5%) and reproducibility (CV: 6%) were obtained with the selected pair of antibodies, with a threshold of positivity at 6 ng/mL. CBA and ELISA techniques were correlated with a Spearman coefficient at 0.99. The reproducibility and reliability of the sCD23 CBA assay were confirmed, with a Spearman coefficient at 0.99 in a series of 23 CLL patients and 13 controls tested in two laboratories equipped with different cytometers and using different lots of CBA reagents. Data obtained with serum and plasma samples were correlated with a Spearman coefficient at 0.99. Our study validates a simple method that allows the clinicians to benefit from an indicator of prognosis at the diagnosis as well as a marker of the evolution of CLL disease.

Soluble CD23 measurement by CBA: A convenient and reliable quantification method in Chronic Lymphocytic Leukemia.

The soluble form of the transmembrane glycoprotein CD23 corresponding to the low-affinity receptor for the immunoglobulin E (sCD23) is found in the serum of patients with chronic lymphocytic leukemia (CLL). In this disease, an increase in sCD23 level is predictive of poor prognosis at diagnosis as well as during clinical outcome. Quantification of sCD23 is classically performed by ELISA assay, a method not routinely used in hematology laboratories. Our aim was to apply cytometric bead array (CBA) technology to measure sCD23 levels. We tested 420 serum samples, 360 from patients and 60 from healthy volunteers. We selected 3 pairs of monoclonal antibodies (moAb) recognizing the CD23 molecule that were tested in various conditions of temperature, centrifugation, washing or chemical supplementation. Satisfactory performances in terms of repeatability (CV: 5%) and reproducibility (CV: 6%) were obtained with the selected pair of antibodies, with a threshold of positivity at 6 ng/mL. CBA and ELISA techniques were correlated with a Spearman coefficient at 0.99. The reproducibility and reliability of the sCD23 CBA assay were confirmed, with a Spearman coefficient at 0.99 in a series of 23 CLL patients and 13 controls tested in 2 laboratories equipped with different cytometers and using different lots of CBA reagents. Data obtained with serum and plasma samples were correlated with a Spearman coefficient at 0.99. Our study validates a simple method that allows the clinicians to benefit from an indicator of prognosis at the diagnosis as well as a marker of the evolution of CLL disease.

Immunohematological reference values for healthy adults in Burkina Faso.

Reference ranges for peripheral blood lymphocyte subsets were generated for 186 healthy adults in Burkina Faso using single-platform flow cytometry. CD4(+) T-cell counts ranged from 631 to 1,696 cells microl(-1); they were lower in males (n = 97) than in females (n = 89), whereas natural killer cell counts were higher.

Evaluation of a simplified dual-platform flow cytometric method for measurement of lymphocyte subsets and T-cell maturation phenotypes in the population of Nouna, Burkina Faso.

In the context of a larger clinical study in Nouna, Burkina Faso, we evaluated a simplified dual-platform (DP) flow cytometric (FCM) method that allows the determination of major lymphocyte subsets in a single test tube. We compared the phenotyping of lymphocytes with DP FCM and simultaneous measurements with standard single-platform (SP) FCM for samples from 177 individuals. Analysis of the comparative measurements revealed that DP FCM systematically underestimates the proportion of NK cells, overestimates the percentage of CD3(+) CD8(+) lymphocytes, and yields proportions of B cells and CD4(+) T cells comparable with the results from SP FCM. Bland-Altman analysis showed a low bias between both methods and an acceptable precision for percent values of CD4(+) T cells (bias +/- precision, -1% +/- 6%) and CD8(+) T cells (-3% +/- 6%). The absolute cell numbers of all lymphocyte subpopulations, however, were systematically biased towards lower values being obtained by DP FCM. Reference values for the distribution of T-cell maturation phenotypes in 177 healthy adults were calculated using DP FCM. The mean +/- standard deviation (SD) CD4(+)-to-CD8(+) T-cell ratio was 1.61 +/- 0.61, the mean percentage +/- SD of CD4(+) T cells was 42% +/- 7%, and that of CD8(+) T cells 29% +/- 7%. Among CD4(+) lymphocytes, 28% +/- 7% were classified as central memory (CD45RA(low) CCR7(+)), 22% +/- 10% as naïve (CD45RA(high) CCR7(+)), 45% +/- 12% as effector memory (CD45RA(low) CCR7(-)); and 5% +/- 3% as terminally differentiated effector memory expressing CD45RA (CD45RA(high) CCR7(-)). Among CD8(bright) lymphocytes, 3% +/- 2% had a central memory phenotype, 27% +/- 13% were naïve, 37% +/- 13% had an effector memory phenotype, and 34% +/- 12% were terminally differentiated effector memory cells expressing CD45RA.