Antibody against mutated citrullinated vimentin: a new sensitive marker in the diagnosis of rheumatoid arthritis.

The aim of this study was to determine the performance of antibodies against mutated citrullinated vimentin (anti-MCV) in comparison with antibodies to cyclic citrullinated peptides (anti-CCP) in patients with rheumatoid arthritis (RA). Serum levels of anti-MCV and anti-CCP were determined in 193 patients with RA and 332 controls, and sensitivity and specificity were calculated. In a separate analysis of 86 patients, the anti-MCV levels were compared to disease activity. Sensitivity of anti-MCV versus anti-CCP was 71.5 and 69.4%, specificity was 81.3 and 97.6%, respectively. The ROC curves showed higher specificity and an advantage of anti-CCP. In seronegative RA patients the sensitivity of anti-MCV was superior over anti-CCP. Anti-MCV positivities also occurred in systemic lupus erythematosus and Sjoegren's syndrome. In a subgroup of 86 RA patients we found a significant correlation between anti-MCV and disease activity. Anti-MCV appears to be an important marker for the diagnosis of RA, and correlates also with disease activity.

Vascular endothelial growth factor in patients with psoriatic arthritis.

OBJECTIVE: To determine the levels of vascular endothelial growth factor (VEGF) in patients with active psoriatic arthritis, patients with inactive psoriatic arthritis, and healthy controls. Serum VEGF levels were correlated with clinical and laboratory features in patients with active psoriasis arthritis. METHODS: Serum samples from 14 patients with active psoriatic arthritis, 14 patients with inactive psoriatic arthritis, and 9 healthy controls were investigated. VEGF levels in the serum were measured using a sensitive sandwich ELISA. RESULTS: The mean serum VEGF concentration in patients with active PA was 394.4 pg/ml (394 +/- 171.8), in patients with inactive PA 200.4 pg/ml (200.4 +/- 115.7), and in healthy subjects 214.3 pg/ml (214.3 +/- 162.1). Patients with active psoriasis arthritis had significantly higher levels of VEGF compared to patients with inactive psoriasis arthritis and healthy individuals (p > 0.001). In contrast, VEGF levels were comparable in patients with inactive psoriatic arthritis and controls (p =0.659). Furthermore, in patients with psoriatic arthritis, VEGF levels were positively correlated with ESR, HAQ, PASI and VAS. CONCLUSION: VEGF levels may be regarded as a good indicator of active psoriasis arthritis.

Lupus anticoagulant and venous leg ulceration.

BACKGROUND: Most leg ulcers occur in patients with venous insufficiency. However, not all patients with venous insufficiency develop leg ulcers. Recent studies have found that factors causing clotting abnormalities, e.g. anticardiolipin antibody (ACA), are associated with leg ulcers. Although lupus anticoagulant, like ACA, belongs to the group of antiphospholipid antibodies, its presence in patients with venous leg ulceration has not been previously reported. OBJECTIVES: To determine the presence of lupus anticoagulant in patients with venous leg ulceration. METHODS: We investigated the presence of lupus anticoagulant in 27 patients with venous leg ulcers and compared these data with controls. Lupus anticoagulant was evaluated in all subjects by the Russell's viper venom test. RESULTS: Of 27 patients with venous leg ulceration, 16 (59%) were shown to have lupus anticoagulant, while only one of 32 controls (3%) was found to have lupus anticoagulant. Thus, lupus anticoagulant was significantly more frequent in patients with venous leg ulcers than in controls (P < 0.001). CONCLUSIONS: We suggest that lupus anticoagulant could be a hitherto unknown factor contributing to the development of venous leg ulcers.

Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics--technique, benefits, limitations and applications.

Autoimmune diseases are relatively frequent disease complexes, affecting approximately five to seven percent of the population. After cardio-vascular and malignant diseases they come third in mortality. As the clinical diagnosis of rheumatic autoimmune diseases is difficult, laboratory tests are helpful in differential diagnosis and for verification of the clinical diagnosis. The most commonly used assay is the determination of ANAs (anti nuclear antibodies) by indirect immunofluorescence (IFA). However, this method lacks reliable standardisation and is very dependable on the qualification of the observer. Enzyme Immunoassays (EIA) and Immunoblotting techniques, on the contrary, attain good standardisation and comparability. However, the latter methods are limited to the presentation of defined autoantibodies only. There is a need to select a suitable strategy for the use of laboratory parameters in order to support the clinical diagnosis more efficiently. A possible strategy is to replace IFA as a first line screening-step by second-generation ANA-EIA kits.

A simple method for measurement of CD14weak CD16strong monocytes 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.

Multicenter evaluation study on a new HEp2 ANA screening enzyme immune assay.

The COBAS Core HEp2 ANA enzyme immune assay (EIA) was evaluated in a precision and a clinical sample study in comparison to indirect immunofluorescence assay (IFA) on HEp2-cells. In the precision study the COBAS Core EIA yielded intraassay coefficient variations (CVs) mostly below 9%, and interassay CVs between 4.7% and 10.4%. When comparing the COBAS Core EIA to IFA, the results corresponded well in healthy subjects, systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis. In the case of Sjögren's syndrome and scleroderma patients the COBAS Core EIA yielded a lower rate of positive results compared to IFA. This discrepancy may be explained by the lack of detection of autoantibodies to nuclear antigens that can be identified only by IFA due to their compartmentalization and higher localized antigen density in HEp2 cells. The discrepancies in the group of dermato/polymyositis patients are due to the fact that the EIA contains mainly nuclear antigens and was able to detect only antibodies against the cytoplasmic Jo1 antigen that was added to the HEp2 nuclear extract. Routine sera were also evaluated; good agreement was found in sera from patients attending tertiary reference centres for autoimmune diseases but a higher number of discrepancies was reported in sera from unselected populations.

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.

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 Wegener's 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.

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.

Value of neutrophil CD16 expression for detection of left shift and acute-phase response.

Fc gamma RIII (CD16) expression of neutrophil granulocytes was measured in 156 patients by means of fluorescence-labeled antibodies with a flow cytometer. Results were compared with (1) 400-cell manual differential count; (2) left shift flagging on hematology analyzers; (3) absolute neutrophil count; and (4) acute-phase protein levels. Asynchrony was noted between neutrophil CD16 expression and microscopically defined neutrophil stage, particularly in heavily left-shifted samples, which made it impossible to reliably enumerate immature neutrophils on the basis of CD16 expression. According to receiver operating characteristics, the absolute count of CD16-negative neutrophils was highly discriminatory for detection of left shift, with an area under the curve (AUC) of 0.842 +/- 0.03 (SE) and maximum efficiency of 81% +/- 3%, but absolute neutrophil count was not significantly inferior (0.821 +/- 0.03 and 76% +/- 3%). STKS and SE9000 flagging demonstrated efficiency of 76% +/- 3% and 81% +/- 3%, respectively. For detection of acute-phase response, absolute neutrophil count (AUC, 0.836 +/- 0.04; maximum efficiency, 80% +/- 4%) outperformed both quantitative neutrophil CD16 expression (0.760 +/- 0.05; 75% +/- 4%) and absolute CD16-negative neutrophil count (0.757 +/- 0.05; 71% +/- 4%); absolute band count performed similarly (0.853 +/- 0.04; 79% +/- 4%) and showed high efficiency at high sensitivity and specificity. Efficiency of analyzer flagging for detection of acute-phase response was not superior to absolute neutrophil count (STKS, 77% +/- 4%; SE9000, 78% +/- 4%). In conclusion, the diagnostic value of measuring neutrophil CD16 expression was generally similar to that of less complicated analytes.

Increased serum levels of soluble CD44-isoform v5 in rheumatic diseases are restricted to seropositive rheumatoid arthritis.

Serum levels of sCD44v5 were measured in 134 patients with definite inflammatory rheumatic diseases (IRD) using a sandwich type ELISA. 94 patients suffered from erosive IgM-rheumatoid factor positive rheumatoid arthritis (RA+), 20 with undifferentiated seronegative polyarthritis, 12 with osteoarthropathia psoriatica and psoriasis vulgaris, 3 with systemic lupus erythematosus, 3 with scleroderma and 2 with reactive arthritis. Elevated serum levels (> 58 ng/ml to 221 ng/ml; median: 93 ng/ml) were only detected in 54/94 (57%) patients with RA+, but not in other IRD. They correlated with advanced stages of disease (Steinbrocker stages III + IV; p < 0.05), elevated CRP-levels (p < 0.01) and higher measurements of IgM rheumatoid factor.

Use of precision profiles to evaluate precision of the automated leukocyte differential.

The commonly used methods of assessing the precision of the automated leukocyte differential have certain drawbacks that affect the validity and comparability of results. In the present report, we introduce a procedure based on building precision profiles from a large number of within-run imprecision experiments. The profiles are fitted to the function for the CV of proportions, which yields the number of theoretically differentiated leukocytes. Differences between fitted curves are evaluated for statistical significance by the F-test. As an example, we compared the precision of two hematology analyzers, a flow-cytometric technique involving fluorescence-labeled monoclonal antibodies, and the manual differential. We were able to establish definite differences in precision between different analyzers and different leukocyte classes. Our data also indicated that conventional within-run imprecision studies may completely misjudge analyzer precision. Furthermore, we could demonstrate that the precision of analyzers that analyze a fixed amount of blood rather than a fixed number of leukocytes is strongly influenced by the leukocyte count of the sample, leading to high imprecision for leukopenic samples. We believe the proposed procedure is a useful addition to currently used protocols; it yields clear results and creates a statistical basis of comparison between various instruments and techniques of differentiation.

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.

HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay.

Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results; one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.

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 II 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 x 10(9) 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.

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; Spearman's 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.

Precision and accuracy of monocyte counting. Comparison of two hematology analyzers, the manual differential and flow cytometry.

The Coulter STKS (Coulter, Hialeah, FL), the Abbott CD3500 (Abbott Diagnostics, Abbott Park, IL), a 400-cell manual differential, and flow cytometry using double-staining with fluorescence-labelled monoclonal antibodies (CD45-FITC and CD14-PE) on a Coulter Epics Profile II were evaluated for precision and accuracy in relative monocyte counting. STKS, CD3500, and Profile II achieved a precision analogous to a 3,542-, 1,835-, and 11,998-cell differential, respectively, demonstrating the superiority of automated methods. Analysis of 156 normal and abnormal samples revealed that the mean relative monocyte counts of the manual differential, CD3500 and Profile II were not significantly different. Only the STKS results showed a positive bias (0.79% +/- 1.65), which was increased in lymphocytic samples. Linear regression between the Profile II as independent viable, and the other techniques yielded acceptable correlation coefficients (STKS: 0.861, CD3500: 0.844, manual differential:0.833). Profile II results were also compared to those of a Becton Dickinson FACScan (Becton Dickinson, Mountain View, CA), which yielded an excellent correlation (r = 0.991) but a slightly smaller relative monocyte count (bias-0.39% +/- 0.60) of the latter. On the basis of these data, the authors recommend the use of monoclonal antibodies as a new reference method, but also indicate the need for further methodological investigations.

Enzymatic determination of sodium, potassium, and chloride in abnormal (hemolyzed, icteric, lipemic, paraproteinemic, or uremic) serum samples compared with indirect determination with ion-selective electrodes.

We evaluated the effect of hemolysis, icteric discoloration, lipemia, paraproteinemia, and uremia on enzymatic methods for determining sodium, potassium, and chloride, according to the National Committee for Clinical Laboratory Standards EP7-P proposals for testing interference from endogenous substances. The sodium, potassium, and chloride assays (reagent kits supplied by Boehringer Mannheim) were based on electrolyte-dependent beta-galactosidase, pyruvate kinase, and alpha-amylase, respectively. The results were compared with those obtained by indirect ion-selective electrodes (ISE), which in turn had been validated by flame photometry. We analyzed the samples with Hitachi 717, 737, and 911 chemistry analyzers and with an IL943 flame photometer. The enzymatic results were in good agreement with those by ISE, the interference-related differences generally being without clinical significance; however, none of the enzymatic methods could analyze grossly lipemic samples.