Peter Michael Bayer

Supplementation with Mixed Fruit and Vegetable Juice Concentrates Increased Serum Antioxidants and Folate in Healthy Adults

Objective: Epidemiological studies have shown that low plasma levels of antioxidant micronutrients, which are commonly found in fruit and vegetables, are associated with increased risk for diseases such as heart disease, cancer, metabolic disorders and the like. The aim of this study was to monitor the dietary habits of a group of healthy, middle-aged, men and women and to assess the effect of supplementation with a natural phytonutrient preparation from fruits and vegetables, on plasma levels of various antioxidant micronutrients and oxidative stress assessed by measuring 8-oxodGuo (8-oxo-7,8-dihydro-2′-deoxyguanosine) in urine. Methods: The study followed a double-blind randomized cross-over design involving 59 healthy men and women (40–60 years of age). The supplement or a placebo was given to two groups for a total period of 14 weeks (crossover week 7). Blood levels of β-carotene, vitamin C, vitamin E, selenium and folate were measured at 0, 7 and 14 weeks. Fruit and vegetable consumption was monitored by means of a retrospective food frequency questionnaire at week 0, 7 and 14. Urinary 8-oxodGuo was also determined at these time points. Results: Significant increases in blood nutrient levels after active supplementation were observed for β-carotene, vitamin C, vitamin E, selenium and folate. Ranges measured, after supplementation, often fell into those associated with a reduced risk for disease. Our data suggests that, although generally health conscious, participants still fell short of the recommended five portions of fruit and vegetables per day. No significant group changes were noted for 8-oxodGuo concentration in urine. Conclusion: Supplementation with mixed fruit and vegetable juice concentrates effectively increased plasma levels of important antioxidant nutrients and folate.

Elevated levels of kappa free light chains in CSF support the diagnosis of multiple sclerosis

BackgroundNumerous studies have demonstrated elevated kappa free light chains (KFLCs) in CSF of multiple sclerosis (MS) patients. However, so far only small cohorts have been examined, and generally only through qualitative KFLCs analysis. Using a recently developed free light chain (FLC) immunoassay, it is now possible to quantitatively measure KFLCs by automated nephelometry. Our objective was to determine the extent to which KFLC levels in CSF correlated with the diagnosis of MS and CISSMS (clinically isolated syndrome suggestive of MS) compared to oligoclonal banding (OCB) and the immunoglobulin G (IgG) index.MethodsCSF and serum samples from 438 unselected patients, including a MS group of 70 patients (41 MS, 29 CISSMS), were analysed using nephelometry and isoelectric focusing. We then retrospectively correlated results with patients’ diagnoses.ResultsOf the MS group (n = 70), 67 patients had elevated KFLCs using the KFLC index (≥ 5.9), 64 patients showed OCB and 56 patients presented with an elevated IgG index (≥ 0.6). Sensitivities were 0.96 for the KFLC index, 0.91 for OCB and 0.80 for the IgG index. The specificity of the KFLC index for the MS group (0.86) was lower than that of OCB (0.92) but distinctly higher compared to the IgG index (0.77).ConclusionIn this study, an elevated KFLC-index represented the most sensitive and specific quantitative diagnostic parameter for MS. As it is measured by automated, routinely available laboratory methods, KFLC quantitation can provide a rapid and reproduceable indication of intrathecal immunological processes supporting current MS diagnostic criteria.

Toward a New Reference Method for the Leukocyte Five-Part Differential

A flow cytometric method performing a five-part leukocyte differential based on three-color staining with anti-CD45-fluorescein isothiocyanate (FITC), anti-CD-14-phycoerythrin (PE)/Cy5, and a cocktail of PE-labeled anti-CD2, anti-CD16, and anti-HLA-DR antibodies was evaluated. Results obtained by using three different sample preparation procedures and two different flow cytometers were compared with those of a 1,000-cell manual differential for evaluation of accuracy. We observed excellent correlations with the manual differential for all leukocyte subclasses and even higher correlations between the different flow cytometric methods. Flow cytometric basophil results were identical to the manual counts, regardless of which sample preparation technique or flow cytometer was used. Therefore, we propose our flow cytometric method as the first acceptable automated reference method for basophil counting. The flow cytometric results for the other leukocyte subclasses were apparently influenced by the sample preparation, which could not be explained by cell loss during washing steps. Moreover, a small influence of the flow cytometer was also observed. Assessing the influence of sample storage, we found only minimal changes within 24 h. In establishing reference values, high precision of flow cytometric results facilitated detection of a significantly higher monocyte count for males (relative count: 7.08 +/- 1.73% vs. 6.44 +/- 1.33%, P < 0.05; absolute count: 0.536 +/- 0.181 x 10(9)/liter vs. 0.456 +/- 139 x 10(9)/liter, P < 0.01). Our data indicate that monoclonal antibody-based flow cytometry is a highly suitable reference method for the five-part differential: It also shows, however, that studies will have to put more emphasis on methodological issues to define a method that shows a high interlaboratory reproducibility.

Proposed reference method for peripheral-blood monocyte counting using fluorescence-labelled monoclonal antibodies.

Flow cytometry using fluorescence-labelled monoclonal antibodies has been proposed as a possible new reference method to evaluate the monocyte counting performance of automated hematology analyzers. Since in previous studies only one such technique was applied, we investigated how different flow cytometric techniques compared to the manual differential and a hematology analyzer. Relative monocyte counts of 60 samples of the daily routine were determined on a Coulter Profile II flow cytometer after incubation with two different CD45-FITC/CD 14-PE antibody combinations and subsequent preparation with two whole-blood lysis techniques, including one no-wash technique. Results were compared to those of a 600-cell manual differential and to those of the Coulter STKS hematology analyzer. All flow cytometric methods correlated very well with the manual differential (r > or = 0.925) and none showed a significant bias. The Coulter STKS relative monocyte counts were slightly higher than those of the manual differential (8.76% vs. 8.18%). The correlations between the methods employing monoclonal antibodies were excellent (r > or = 0.995) and the mean monocyte counts identical although a small, non-systematic influence of sample preparation techniques was noted. An influence of the antibody clones was not observed. The precision of the Profile II results was far superior to that of the manual differential and the STKS. Our data show that flow cytometry employing fluorescence-labelled monoclonal antibodies is a potentially ideal new reference method for monocyte counting. However, they also show that establishing a new reference method will require extensive investigation and exact definition of the sample preparation procedure to be used.

Differential expression of tumor necrosis factor receptor subtypes on leukocytes in systemic inflammatory response syndrome.

OBJECTIVE To determine the expression of tumor necrosis factor (TNF) receptor in patients with systemic inflammatory response syndrome (SIRS). DESIGN Prospective study. SETTING Intensive care unit and central laboratory. PATIENTS Blood specimens from 18 healthy volunteers (controls) and 16 patients with SIRS. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Using monoclonal antibodies, fluorescence labeling, and high sensitivity flow cytometry, we measured the expression of membrane TNF receptor subtypes TNF-R55 and TNF-R75 on peripheral blood leukocytes. Receptor expression is expressed as mean fluorescence intensity +/- SD (units: detection channel number). In controls, TNF-R55 was only weakly expressed (monocytes: 2.5+/-1.8; neutrophils: 0.7+/-0.8), whereas expression of TNF-R75 was higher (monocytes: 28.6+/-9.0; neutrophils: 4.8+/-1.0) and was also found on lymphocytes (on CD8+ lymphocytes: 5.7+/-1.8; CD16+: 5.5+/-1.2; CD4+: 9.7+/-3.7). In SIRS, we observed increased expression of TNF-R55 on monocytes (6.9+/-3.4, p<.001) and neutrophils (2.2+/-1.9, p<.01), as well as decreased expression of TNF-R75 on monocytes (17.3+/-13.2; p<.001). The extent of TNF-R55 up-regulation did not correlate with that of TNF-R75 down-regulation. TNF-R55 on monocytes and neutrophils strongly correlated with body temperature but not with survival, whereas monocyte TNF-R75 was considerably lower in nonsurvivors, albeit not significantly (12.3+/-7.1 vs. 23.9+/-16.7; p = .07). CONCLUSIONS These data indicate that leukocyte TNF-R55 and TNF-R75 react differentially and probably serve different functions in SIRS, which prompts the investigation of receptor subtype-specific therapeutic approaches.

Peripheral blood monocyte counting : towards a new reference method

Flow cytometric enumeration of monocytes stained with fluorescence-labelled monoclonal antibodies has been proposed as a possible reference method for monocyte counting. We compared precision and accuracy of monocyte counting of the Coulter STKS, the Cobas Argos 5 Diff, the 800-cell manual differential, and the Coulter Epics Profile II flow cytometer using double-staining with fluorescence-labelled monoclonal antibodies (CD45-FITC and CD14-PE). Precision: STKS, Argos and Profile II achieved a precision analogous to a 3423-, 1298-, and 11089-cell differential, respectively, confirming the superiority of automated methods. Accuracy (136 normal and abnormal samples): Correlation of automated methods with the manual differential was good (STKS: r = 0.934, Argos 5 Diff: r = 0.808, Profile II: r = 0.924; Spearmans rank correlation coefficient). The mean relative STKS monocyte result was 0.52 +/- 1.63% (mean +/- SD) higher than the manual differential, whereas the Argos 5 Diff results were 1.22 +/- 2.51% lower (p < 0.001). Profile II results showed a small bias against the manual differential (-0.18 +/- 1.44%, p < 0.05). Analysing 135 healthy adult subjects on the Profile II, males were found to have a higher mean monocyte count (relative count: 6.95 +/- 1.43% vs. 5.86 +/- 0.98%; absolute count: 0.48 +/- 0.15 x 10(9)/l vs. 0.39 +/- 0.11 x 10(9)/l, p < 0.001) and a higher and wider normal range than females (relative count: 4.97 to 9.78% vs. 4.26 to 7.81%, absolute count: 0.30 to 0.84 x 10(9)/l vs. 0.25 to 0.65 x 10(9)/l). Flow cytometry based on fluorescence-labelled monoclonal antibodies for monocyte enumeration seems an efficient tool to evaluate the monocyte counting performance of haematology analysers and an ideal successor to the manual differential as reference method for monocyte counting.

Anticytokeratins are a potential source of false‐positive indirect immunofluorescence assays for C‐ANCA

Antibodies to neutrophil cytoplasmic antigens (ANCA) targeted toward granule enzymes have been recognized as a valuable diagnostic tool in the detection of Wegeners granulomatosis and systemic vasculitides. However, the most commonly used method of detection, the indirect immunofluorescence assay, is prone to false‐positive results due to antibodies of different pathological significance either targeted to, or cross‐reacting with, similarly distributed epitopes. Using double immunofluorescence, the present study demonstrates that anticytokeratin antibodies are able to produce false‐positive C‐ANCA immunofluorescence assays. In addition, a case of natural appearance of cytokeratin‐reactive antibodies causing a false‐positive “pseudo‐ANCA” staining pattern in a patient presenting with sepsis is reported. Since the expression of cytokeratins is almost exclusively confined to epithelial cells, the most plausible explanation for both phenomena is a crossreaction of anticytokeratin antibodies with granule associated epitopes. Due to the natural appearance of anticytokeratin antibodies in association with a variety of other pathologic entities, it is of crucial importance for the diagnostic significance of the C‐ANCA immunofluorescence assay to exclude anticytokeratin caused false‐positive results. It is shown that supplementary indirect immunofluorescence tests performed on cultured human epithelial cells readily distinguish anticytokeratin caused “pseudo‐ANCA” from true C‐ANCA. J. Clin. Lab. Anal. 12:54–59, 1998.

Evaluation of automated basophil counting by using fluorescence-labelled monoclonal antibodies.

The shortcomings of current methods of basophil enumeration detract from the clinical value of the basophil count. Moreover, sophisticated and costly techniques of automated basophil counting hardly can be validated for lack of a suitable reference method. We investigated whether a flow cytometric technique using double staining with fluorescence‐labelled monoclonal antibodies (mAb) CD45‐FITC and CD14‐PE on a Coulter Epics Profile II could be used to evaluate basophil counting performance of hematology analyzers. The technique was compared with the 800‐cell manual differential, the Coulter STKS, and the Cobas Argos 5 Diff. Precision: STKS, Argos and Profile II showed a precision analogous to a 2,173, 2,250‐, and 14,705‐cell differential, respectively, illustrating the superiority of automated methods. Accuracy (150 normal and abnormal samples): Using the Profile II as reference the STKS showed a notably weaker correlation than the Argos (r = 0.581 and 0.718, respectively), although this difference was nearly concealed when the imprecise manual differential served as reference (r = 0.517 and 0.562, respectively). The Profile 11 correlated relatively well with the manual differential (r = 0.730). Analyzing 137 healthy adult subjects, we obtained a reference range of 0.33 to 1.35% (0.020 to 0.102 × 109, basophils/L) for the mAb‐based method. These data would recommend mAb‐based basophil counting as a valuable tool for instrument evaluation. However, an observed bias of 0.09% against the manual differential suggests that modifications are necessary before this technique can be considered as new reference method.

A Simple Method for Measurement of CD14weakCD16strongMonocytes in Peripheral Blood

Mounting evidence for the clinical significance of the CD 14weak CD16strong monocyte subpopulation in peripheral blood induced the demand for an efficient method for its determination. We propose a simple, fast, no-wash flow cytometric method using fluorescence-labelled anti-CD14, anti-CD16, and anti-HLA-DR antibodies and ammonium chloride-based erythrocyte lysis. This type of analysis can be performed on a standard three-colour flow cytometer. The method avoids interference by NK-cells and neutrophil granulocytes without defining monocytes by stringent light scatter criteria that might lead to a loss of CD14weak CD16strong monocytes. It, therefore, offers high reliability and accuracy. Its performance recommends the method to be used for routine clinical measurements of CD14weak CD16strong monocytes.

Adenylate Kinase Activity of Cerebrospinal Fluid in Central Nervous System Disorders

Adenylate kinase activity was measured in 41 samples of cerebrospinal fluid in 34 patients with various neurological disorders or psychiatric symptomatologies. Activities of the enzyme showed to be linked to clinically estimated acuteness or progression of the changes in the central nervous system at the time of specimen collection. The findings suggest the conclusion that determination of adenylate kinase activity in cerebrospinal fluid is a meaningful tool for the evaluation of progression and/or acuteness of central nervous system disorders.