Comparison of the clinical utility of the Elia CTD Screen to indirect immunofluorescence on Hep-2 cells.

BACKGROUND: We compared the Elia CTD Screen (ECS), a fluoroenzymeimmunoassay incorporating 17 human antinuclear antigens (ANA), with indirect immunofluorescence (IIF) on Hep-2 cells in order to determine the clinical utility of the ECS in additon to or without IIF. METHODS: We examined 1708 consecutive serum samples submitted for ANA testing using the ECS and IIF in parallel. Positive screen results were further examined by quantitative fluoroenzymeimmunoassays and/or immunoblots for antibody identification. The medical records were evaluated for systemic rheumatic disorders. RESULTS: Concordance between ECS and IIF was observed in 1344 (78.8%) samples. ECS had a better detection rate for anti-dsDNA, -SSA/Ro, -SSB/La, -U1RNP and -Jo-1 antibodies, whereas IIF was superior in the detection of anti-CENP-B antibodies as well as anti-histone, -nucleosome and -Pl-12 antibodies, which are not included in the ECS antigen panel. ECS had a 100% sensitivity for Sjögren's syndrome, systemic sclerosis and Sharp syndrome. The sensitivity for Sjögren's syndrome was slightly higher for ESC than for IIF (94%). IIF had a higher diagnostic sensitivity for systemic lupus erythematosus, indeterminated connective tissue disease, Raynaud's syndrome and limited scleroderma, compared to ESC (100% vs. 80%, 100 vs. 75%, 89 vs. 57%, 100 vs. 88.9%). CONCLUSIONS: Our results suggest that the ECS represents an appropriate diagnostic tool for ANA screening. However, since some antigens are not incorporated in the ECS panel, and some ANA can also be missed by IIF, sequential or parallel screening with ECS and IIF may be reasonable when the clinical suspicion for connective tissue disease is high.

Cytospin preparations are superior to common smears in the detection of monosodium urate crystals in low-cellular synovial fluids.

In cases of gout with a low synovial fluid (SF) leukocyte count and atypical clinical presentation, such as in intercritical periods, the load of monosodium urate (MSU) crystals is frequently low, and thus, methods to improve the crystal detection may be beneficial. We compared the MSU crystal detection rates between cytospin slides and common smear preparations in low-cellular (<2,000/µl) SF samples of patients with gout. We determined the number of MSU crystals/15 high power fields (HPF) at × 1,000 magnification by polarised microscopy in cytospin preparations and smears in SF samples of 17 patients with MSU-crystal-proven gout and compared the two methods statistically. All of the cytospin slides (100 %) contained MSU crystals and showed a median number of 124 crystals/15 HPF (range 2-3,915), whereas 5 of the 17 smears (29 %) were MSU-crystal-negative, with a median count of 2 crystals/15 HPF (range 0-430/HPF). The difference was statistically highly significant (p < 0.0001). In conclusion, we have shown that the cytospin technique is superior to smears in the detection of MSU crystals in SF with a low SF leukocyte count. In light of our observations, we recommend the use of cytocentrifuges for routine crystal analysis in such cases.

Assessment of the response to acetylsalicylic acid in patients with myeloproliferative neoplasms by whole blood assays: a comparison of the PFA-100 with multiple electrode aggregometry.

Since thrombotic and haemorrhagic complications are the most important causes of morbidity and mortality in myeloproliferative neoplasms (MPN), establishing valid techniques for the monitoring of antiaggregatory treatment would be beneficial. The aim of this study was to assess the aspirin responsiveness in patients with MPN by multiple electrode aggregometry (MEA) and the PFA-100, to determine the concordance rate between the two techniques and to examine a potential clinical impact. Twenty-two consecutive outpatients with polycythaemia vera and essential thrombocythaemia receiving long-time treatment with 100 mg of aspirin were included and clinically re-evaluated within six months after study entry. All subjects were identified as aspirin responders using the PFA-100, whereas only nine (41%) study participants were detected as responders by MEA. The difference in the response rates was statistically highly significant (p < 0.0001). The median aggregation result was 55.5 U (8-123) in the ASPI test, and the median PFA-100 closure time (CT) was 300 sec (221 to 300) in the COL-EPI test. Within the clinical observation period no thrombotic or haemorrhagic events occurred in the study population. In this study we concluded that MEA and the PFA-100 are suitable devices for the detection of a response to aspirin treatment in patients with MPN, but differ significantly in the response rates and thus show a low concordance rate.

The detection of calcium pyrophosphate crystals in the synovial fluid of patients with rheumatoid arthritis using the cytospin technique: prevalence and clinical correlation.

There are only a few studies dealing with the detection and clinical impact of calcium pyrophosphate (CPPD) crystals in patients with rheumatoid arthritis (RA) published to date. In particular, data determined by the cytospin technique, which is an effective tool to enhance the crystal detection rate, are lacking. The objectives of this study were to determine the prevalence of CPPD crystals in the synovial fluid (SF) of patients with RA and to investigate whether the detection of CPPD crystals is correlated with demographic, clinical and serological features. We examined 113 consecutive SF samples of patients with RA, obtained from therapeutic arthrocentesis of knee joints. After cytocentrifugation, the sediments were examined by polarized microscopy for the occurrence of CPPD crystals. Demographic, clinical and serological data, acquired from the medical records, were compared between crystal-positive and crystal-negative subjects. CPPD crystals were observed in 20 of the 113 cases, representing 17.7%. CPPD-positive and CPPD-negative subjects did not differ significantly in sex, duration of disease, Steinbrocker radiologic stage, disease activity score 28, as well as serum rheumatoid factor and anti-CCP positivity. Patients positively tested for CPPD crystals had a significantly higher age than CPPD-negative patients (p < 0.0001). An age-independent association of long-time treatment with diuretics and CPPD crystal formation was not found. In conclusion, demographic, clinical and serological characteristics of patients with RA were not associated with the occurrence of CPPD crystals. Age was the only significant influencing factor on CPPD crystal formation in patients with RA.

The cytospin technique improves the detection of calcium pyrophosphate crystals in synovial fluid samples with a low leukocyte count.

In synovial fluids (SF) with low leukocyte or/and crystal counts, important features may be missed, if exclusively smears are examined by polarized microscopy. That may be overcome by cytocentrifuges, which use low-speed centrifugal force to concentrate cells onto a glass slide and thus enhance the number of cells per high power field (HPF). We compared the calcium pyrophosphate (CPP) crystal counts in cytospin preparations with those in common smears of SF. The number of CPP crystals was counted in 50 SF samples by polarized microscopy, and statistical comparisons of the mean values of the cytospin and smear preparations were performed using the Wilcoxon test. The reproducibility within the slides of the cytocentrifuge and smear samples was determined by Spearman's rank correlation. The crystal counts were significantly higher in the cytospin than in the smear preparations (median 96/10 HPF vs. 2.5/10 HPF, p < 0.0001). The correlation in the crystal count between the slides 1 and 2 was significantly higher within the cytocentrifuge than in the smear group (0.97 vs. 0.73, p = 0.0004). CPP-negative cytospin preparations in initially smear-positive slides were not observed. We confirmed that the cytospin technique significantly enhances the number of examinable crystals per HPF, compared to common smears.

Changes in response to antiaggregatory treatment in patients with myeloproliferative neoplasms: a sequential study using multiple electrode aggregometry.

In the present study, we used multiple electrode aggregometry (MEA) to investigate the response to aspirin and clopidogrel treatment, and its potential changes over a long-time disease course in patients with myeloproliferative neoplasms (MPNs). arachidonic acid (ASPI), ADP, and thrombin receptor activating peptide (TRAP) tests were performed at two timepoints between 32-50 months in 21 patients with MPN and 1-46 months in 29 controls. We further checked the medical records of the participants to identify a potential correlation of changes in the treatment response with clinical events. In MPN, four out of 13 patients treated with 100 mg of aspirin, no patients receiving 50 mg of aspirin, and one out of five clopidogrel-treated patients showed a therapeutic antiplatelet effect. In the subsequent examinations, five patients changed from response to nonresponse or vice versa. Initial nonresponse and changes from an initial response to nonresponse were observed in six patients with thrombotic events. In the controls, 25 out of 26 aspirin-treated patients and two out of three clopidogrel-treated patients showed an initially adequate in-vitro response. Except from one patient changing from initial aspirin nonresponse to response, all controls showed a stable response state. One control with two ischemic strokes showed a nonresponse to clopidogrel. In conclusion, MEA detects the response to antiaggregatory treatment, as well as its changes during the disease course in patients with MPN. An initial or subsequent nonresponse was observed in patients with thrombotic events.

The detection of calcium pyrophosphate crystals in sequential synovial fluid examinations of patients with osteoarthritis: once positive, always positive.

The aim of this study was to investigate whether calcium pyrophosphate dihydrate (CPPD) crystals are constantly detectable in sequential synovial fluid (SF) examinations of patients with initially CPPD-positive osteoarthritis (OA). For this purpose, we searched our SF database for CPPD-positive patients, who had two or more SF analyses between 2008 and 2012 to get sequential information. The database contains SF data determined by a standardised procedure. Of 1,878 samples, examined within the defined time period, 60 samples from sequential SF analyses of 23 patients were eligible for this study. The number of examinations ranged from 2 to 7 (median, 2), and the median interval between the first and last arthrocentesis was 12 months (2-43). CPPD crystals were detected in all of the sequentially examined samples according to the defined criterion of positivity. All of the subjects had OA of the knee, with a median Kellgren-Lawrence grade of 3 in the initially performed X-rays, and meniscal calcifications were found in six subjects. In conclusion, our results suggest that CPPD crystals are a regular feature of sequentially examined SF of initially CPPD-positive patients with symptomatic OA of the knee.

Dried cytospin preparations of synovial fluid are a stable material for long-time storage and delayed crystal analysis.

The aim of this study was to evaluate dried SF cytospin preparations as a suitable medium for long-time storage and delayed crystal analysis. For this purpose, we analyzed ten MSU-positive, ten CPPD-positive and 20 crystal-negative SF at baseline (wet preparation), after 24 h, 1 week, 4 weeks, 6 months and 12 months for the occurrence of crystals. After cytocentrifugation for 10 min at 700 rpm in a Shandon Cytospin 4 cytocentrifuge (Thermo Fisher Scientific, Waltham, USA), the sediments were dried on the slides and examined in blinded fashion at any time point by an experienced analyst using polarized microscopy. The crystal content of the initially MSU- and CPPD-positive samples was positively confirmed at any time point of the study, whereas the controls remained crystal-negative during the whole study period. Thus, compared to the examined wet preparations at baseline, there were no false positive or false negative results observed. In conclusion, dried cytospin preparations were confirmed as a suitable material for long-time storage and delayed crystal identification.

Pseudoeosinophilia of synovial fluid is caused by crystals mimicking the distinct light scattering fractions of eosinophilic granules.

BACKGROUND: Automated leukocyte differential counts of synovial fluid (SF) can be influenced by laboratory artefacts. Pseudoeosinophilia of SF has recently been first described in association with monosodium urate and calcium pyrophosphate crystals. This study compared automated measurements of the percentages of SF leukocyte fractions by two haematology analysers in order to elucidate the underlying mechanism of pseudoeosinophilia. METHODS: The percentages of the leukocyte fractions of 17 crystal-containing and 28 crystal-free specimens were compared using the Wilcoxon test. Measurements were performed using the Cell-Dyn 3200 and the ADVIA 2120i, which are based on different techniques. RESULTS: The percentages of eosinophils of the crystal-positive samples determined by the Cell-Dyn 3200 were significantly higher than those assessed by the ADVIA (p<0.0001), whereas the percentages of eosinophils of the controls did not differ significantly between the two devices (p=0.95). The Cell-Dyn 3200 clearly showed the phenomenon of crystal-associated pseudoeosinophilia (p<0.0001), which did not occur in the ADVIA measurements (p=0.28). The percentage of neutrophils was to a lower degree elevated in the crystal group (p=0.015). CONCLUSIONS: It was confirmed that SF crystals interfere with the typical light scattering fractions of leukocyte granules and may thus lead to spuriously elevated percentages of eosinophils and neutrophils in SF specimens.

Calcium pyrophosphate and monosodium urate crystals in synovial fluid as a cause of pseudoeosinophilia.

BACKGROUND: Synovial fluid (SF) leukocytes can be counted microscopically in a Neubauer chamber or by automated procedures using haematology analysers. Knowledge of laboratory artefacts is crucial for the correct interpretation of results obtained using automated methods. SF pseudoeosinophilia has recently been described as a new artefact in patients with crystal-related arthropathies. We investigated whether pseudoeosinophilia of SF is restricted to crystal-related disorders, or if it may also occur in other arthropathies. METHODS: We compared the percentages of eosinophils in 120 crystal containing SF samples with 185 crystal-free specimens using the Wilcoxon test. Crystal positive samples, determined by polarised microscopy, contained at least two monosodium urate or calcium pyrophosphate crystals per 10 high power fields (630× magnification). True SF eosinophilia was ruled out by microscopic examination of stained slides. RESULTS: Crystal positive samples had significantly higher percentages of eosinophils than the controls (p<0.0001). No significant differences between the two crystal types were found (p=0.693). Thus, pseudoeosinophilia was significantly correlated with the presence of crystals, and none of the distinct crystal types was more likely to be associated with pseudoeosinophilia. CONCLUSIONS: In this study, SF pseudoeosinophilia was confirmed as a crystal-related laboratory artefact which has to be considered in the interpretation of automated SF leukocyte differential counts.

Ultra fast liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of cyclosporin A, tacrolimus, sirolimus, and everolimus in whole blood using deuterated internal standards for cyclosporin A and everolimus.

Specific chromatographic methods for the measurement of cyclosporin A, tacrolimus, sirolimus, and everolimus blood levels in patients with organ transplants are time consuming when large numbers of samples must be processed. The authors developed a robust and fast (1 minute) online solid-phase extraction liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of cyclosporin A, tacrolimus, sirolimus, and everolimus. After protein precipitation of the whole blood with zinc sulphate and methanol, the supernatant was loaded on a wide pore reversed-phase column and cleansed of potential interferences with high flow for 20 seconds. After column switching, the analytes were transferred within 20 seconds in the back-flush mode to a short phenyl-hexyl column. The valve was then returned to its initial position and the chromatographic separation performed within 20 seconds. In the meantime, the loading column was prepared for the next injection. Ammoniated adducts of protonated molecules were used as precursor ions for all analytes. Multiple-reaction mode transitions for each immunosuppressant and the internal standards were used for quantification. The working range of the method was 10-1500 microg/L for cyclosporin A, 1.0-44 microg/L for tacrolimus, 1.0-48 microg/L for sirolimus, and 1.2-48 microg/L for everolimus. Within and between-run assay coefficients of variation ranged from 1.8% to 13.0%. The described liquid chromatography/tandem mass spectrometry method shows best performance using the internal standards cyclosporin A-d4 for cyclosporin A, everolimus-d4 for everolimus and ascomycin for tacrolimus and sirolimus. In conclusion, the authors present a very fast, robust, and economical analytical method for therapeutic monitoring of multiple immunosuppressants in daily clinical practice.

Psychological and systemic stress reactions of patients during hyperthermia treatments.

PURPOSE: This study evaluated psychological stress reactions during hyperthermia (HT) treatments and compared them to systemic stress reactions. MATERIALS AND METHODS: 27 patients with malignant disease were treated with superficial or regional hyperthermia. Cortisol and the catecholamines adrenaline and noradrenaline in venous blood were used as markers of psychological stress. The anxiety proneness of the patients was evaluated with a trait-anxiety inventory. Blood pressure and heart rate were markers of systemic stress. Patients were first grouped by superficial or regional treatment and then into two subgroups: anxious and non-anxious patients. RESULTS: The time course of the cortisol concentration of the superficial group showed a slight but significant decrease and that of the regional group an increase. The cortisol concentration of the regional group was sometimes slightly but significantly higher than in the superficial group, and in the group of anxious patients higher than in the group of non-anxious patients. The changes in time courses and differences in groups were not as pronounced for the catecholamine concentrations. Heart rate and blood pressure showed a significant increase only in the regional group, and there was no significant difference between the regional and the superficial groups. None of the variables showed a significant difference between the initial and the subsequent treatments; all lay well within the normal physiological range. CONCLUSIONS: These standard hyperthermia treatments are not excessively stressful, either systemically or psychologically. The different behaviours of the variables confirm that it makes sense to consider systemic stress as well as psychological reactions.

Determination of myeloperoxidase in EDTA plasma: comparison of an enzyme-linked immunosorbent assay with a chemiluminescent automated immunoassay.

BACKGROUND: The increased demand in clinical chemistry laboratories for determination of myeloperoxidase (MPO) as a potential marker for cardiovascular diseases has led to the development of several analytical methods, especially enzyme-linked immunosorbent assay (ELISA, e. g. from Immundiagnostik AG), and recently a new chemiluminescent automated immunoassay (CMIA, Architect MPO assay, Abbott Diagnostics). METHODS: We compared data from 115 patients obtained from an ELISA reference method (Immundiagnostik AG) with data obtained from the automated Architect MPO assay. RESULTS: The new MPO assay from Abbott Diagnostics correlates well with the ELISA (y=0.767x+7.035; R(2)=0.9204). The difference plot according to Bland-Altman analysis shows a mean bias of -1.048 microg/l, with a probability of 95%. CONCLUSIONS: The Architect MPO assay can easily be adapted to the Architect instrument system and it is advantageous as far as technology and analysis time are concerned. This new automated MPO assay shows excellent analytical performance and provides a precise and convenient automated method for the measurement of MPO.

Myeloperoxidase as serum marker for detection of CMV infections and rejections in patients after liver or heart transplantation.

Rejection episodes and infections are common problems after organ transplantations (TX). Rejection can be diagnosed in liver-transplant (LTX) patients when liver-specific enzymes in the serum are elevated. As endomyocardial biopsy (EMB) is the gold standard for detecting heart transplant (HTX) rejection, serum parameters would permit more selective use of this invasive procedure. Cytomegalovirus (CMV) infections can have serious consequences for TX patients and so should be diagnosed and treated timely. At present, there are no suitable diagnostic methods other than CMV antigen pp65 and CMV polymerase chain reaction (PCR). Our study aimed to test the sensitivity of myeloperoxidase (MPO), an enzyme of neutrophilic granulocytes, as a new serum parameter in addition to established serum parameters and EMB for diagnosis of infection and rejection episodes after LTX and HTX. MPO in plasma from 246 blood samples (103 used for statistical analysis) from 27 patients (18 LTX and 9 HTX) was determined using ELISA; C-reactive protein (CRP), gamma-glutamyl-transpeptidase (GGT), white blood count and CMV pp65 antigen were monitored routinely. EMBs were performed at defined intervals after HTX. Results were analyzed with descriptive statistics, T-test, Wilcoxon test and Cox regression analysis, whereby a p<0.05 was viewed as significant. MPO values in TX patients with an infection (7 LTX, 2 HTX) were significantly higher than in TX patients without complications (control group) (253.9 microg/l vs. 116.6 microg/l, p=0.0194). In TX patients with rejections (6 LTX, 6 HTX), there is also a significant increase in comparison to controls (429.7 microg/l vs. 116.6 microg/l, p=0.0001). Data from individual TX patients, however, indicate that MPO levels rise distinctly earlier with infection (CMV) than with rejection, enabling earlier detection of the complication and initiation of suitable treatment. Our findings suggest that a larger and prospective study should be designed to evaluate the usefulness of MPO levels in assessing organ transplant recipients.

Drugs and brain death diagnostics: determination of drugs capable of inducing EEG zero line.

BACKGROUND: Several drugs may affect the diagnosis of brain death by depressing the electroencephalographic signal. Serum levels of these drugs must be below their respective therapeutic ranges. METHODS: A high performance liquid chromatography-based fast and simple method was developed for determination of thiopentone, pentobarbitone, phenobarbitone, methohexital and propofol in serum and validated according to international recommendations. RESULTS: Separation of extracted analytes was performed on a reversed phase column [Agilent Zorbax SB C18, 5 microm, 4.6 x 250 mm; mobile phase 50% 50 mM NaH(2)PO(4) pH 4.6 mixed with 35% (v/v) acetonitrile and 15% (v/v) methanol]. Calibration curves were linear throughout the selected ranges (microg/mL, thiopentone 0.25-50, pentobarbitone 0.25-25, phenobarbitone 2.5-50, methohexital 0.125-2.50, propofol 0.25-5.0). The standard deviations for the regression line, recovery, imprecision and accuracy results were all highly satisfactory. The lower limits of quantification for propofol, thiopentone and pentobarbitone were set at 0.25 microg/mL, for phenobarbitone 2.5 microg/mL, and for methohexital 0.125 microg/mL, which are below the lowest pharmacologically relevant serum concentrations. Intra- and inter-day coefficients of variation were less than 10% throughout as determined with six replicates. CONCLUSIONS: The method presented is suitable for drug monitoring to help enhance the reliability of the diagnosis of brain death.

Comparison of fluorescent polarization immunoassay (FPIA) versus HPLC to measure everolimus blood concentrations in clinical transplantation.

Clinical management of transplant patients depends on therapeutic drug monitoring (TDM) and regulation of immunosuppressive therapy. TDM of whole-blood concentrations is mandatory for everolimus (ERL) dosage individualisation. We compared the new semi-automated immunoassay (Innofluor Certican Assay System, Seradyn Inc) using FPIA technology on Abbott TDxFLx analyzers with established HPLC-UV as reference method. A total of 165 samples were analyzed from 52 transplant patients (40 kidney, 12 heart) receiving ERL or another immunosuppressive agent as part their routine care after organ transplantation. The correlation coefficient was r(2)=0.8229, and the regression equation (95% IC) yielded FPIA=1.111 x (HPLC)+0.378. FPIA compared to HPLC gave a positive bias of 1.19 ng/ml. The FPIA assay so appears to have a diagnostic efficacy comparable to HPLC for assessing the risk of acute rejection in transplant recipients. However, the values of the FPIA were higher than those calculated from HPLC measurements, because of the cross-reactivity of the antibody used in the FPIA assay with the ERL metabolite and/or with sirolimus; this cross-reactivity occurs frequently when transplant patients are switched from sirolimus to ERL.

Determination of cyclosporine A in whole blood: comparison of a chromatographic method with three different immunological methods.

Cyclosporine A is potent immunosuppressive agent characterized by a narrow therapeutic range, inter- and intra-individual variability and a lack of correlation between drug dosage and blood levels. In view of these facts, blood levels of CyA should be routinely monitored to assess organ rejection and toxicity. We evaluated CyA as well as its metabolites (AM9, AM19, AMl, and AM4N) in whole blood samples from 117 patients using commercially available immunological assays (AxSYM, EMIT, Dimension) and HPLC. Cross-reactivity of the immunological assays was evaluated using different concentrations of the CyA metabolites (in vitro cross-reactivity) and by statistical analysis of patient data (in vivo cross-reactivity). Cross-reactivity was seen in all immunological assays, with differences in in vitro and in vivo cross-reactivity. The statistical analysis showed a classical correlation between HPLC and AxSYM of r(2)=0.89, HPLC versus EMIT of r(2)=0.93, and HPLC versus Dimension of r(2)=0.93. The percentage metabolite cross-reactivity (%) by immunological assays for four metabolites at two concentrations each (250 and 1000 ng ml(-1)) was lowest with the Dimension assay. Of the immunological methods examined, the new Dimension for CyA determination can be relied on to produce results comparable to HPLC; other advantages are its simplicity, practicability and ease of handling.