[The diagnostic accuracy of anti-citrullin antibody assessment in the diagnosis of patients suspected of rheumatoid arthritis by a general practitioner]. / De diagnostische accuratesse van de anticitrullinebepaling voor het vaststellen van reumatoïde artritis door de huisarts.

OBJECTIVE: To determine whether assessment of antibodies directed against citrullin provides additional value in the diagnosis of rheumatoid arthritis (RA) in general practice. DESIGN: Retrospective. METHODS: In a 6-month period in 2004 (May-December), all sera sent to our laboratory for assessment of rheumatoid factor (RF-IgM), were also analysed for the presence of antibodies directed against citrullinated fibrinogen (anti-citrullin). We analysed 691 sera sent in by general practitioners using a homemade assay. To determine the disease classification, general practitioners were asked to provide information pertaining to the American College of Rheumatology disease classification criteria. The response was 97.6%. For patients who were referred to a rheumatologist in the last 2 years (December 2004-December 2006), the diagnosis of the rheumatologist was also considered in the analysis. RESULTS: A total of 28 patients (4%) were diagnosed with rheumatoid arthritis. Only 25% of these patients were positive for anti-citrullin, and only 25% were positive for RF-IgM. These 2 groups only partially overlapped. The positive and negative predictive values of anti-citrullin were 36 and 96%, respectively. CONCLUSION: The presence of anti-citrullin provided no additional value compared to rheumatoid factor in classifying RA in a general practice population.

A novel time resolved fluorometric assay of anoikis using Europium-labelled Annexin V in cultured adherent cells.

BACKGROUND: Adherent cells undergo apoptosis when detached from their home ground, a process called anoikis (homelessness). METHODS: We developed a new and sensitive method to analyse apoptosis and anoikis of adherent cell types using a time resolved fluorometric assay with Europium-labelled Annexin V. Anoikis was induced with tumor necrosis factor-alpha/cycloheximide and three cell fractions of the cell cultures were prepared and analysed. Fraction 1 consisted of adherent cells, analysed while growing on their support (without detachment by trypsinisation). Fraction 2 contained detached cells due to anoikis (floating cells) and fraction 3 contained apoptotic bodies. Both fractions 2 and 3 were present in the culture medium and were isolated by differential centrifugation. RESULTS: TNF-alpha treatment of three different types of adherent cell cultures induced a significant increase of the amount of floating cells (anoikis) and apoptotic bodies compared to control cell cultures. Also in the adherent cell fractions a small amount of apoptosis was observed. CONCLUSIONS: The novel time resolved assay provides the ability to analyse the cell death cascade in adherent cell cultures of the same sample at the same time in a sensitive and reproducible way.

Rheumatoid factor measured by fluoroimmunoassay: a responsive measure of rheumatoid arthritis disease activity that is associated with joint damage.

OBJECTIVES: To determine whether rheumatoid factors (RFs), measured as continuous variables by time resolved fluoroimmunoassay, reflect disease activity in rheumatoid arthritis (RA). Further, to study the association of RFs and other disease activity parameters with radiological joint damage, especially in individual patients. METHODS: In active, early RA, IgM and IgA RFs, as well as erythrocyte sedimentation rate (ESR), C reactive protein (CRP), tender joint score, and swollen joint score were assessed regularly. At the study start and at 56 and 80 weeks, radiographs of hands and feet were assessed by the Sharp score (van der Heijde modification). Associations between RFs and disease activity parameters were studied. In addition, associations between radiographic damage and disease activity parameters (baseline and time integrated) were analysed by non-parametric tests and multiple regression analysis. The relation between time integrated disease activity parameters and radiological damage in individual patients was analysed and visualised. RESULTS: 155 patients were included. RF levels were strongly associated with the disease activity parameters (especially ESR and CRP) and with each other. All disease activity parameters, at baseline as well as time integrated parameters, were associated with (the progression of) radiographic damage. Moreover, in individual patients, a linear relationship between time integrated disease activity parameters and progression of radiological damage was seen. CONCLUSION: RFs, measured as continuous variables, can be considered as disease activity parameters in patients with RA. The level of RF at baseline and the exposure to RF over time is associated with radiological damage. In individual patients, there is a constant relation between disease activity and radiological damage.

The use of free cortisol index for laboratory assessment of pituitary-adrenal function.

We developed a time-resolved-fluoro-immunoassay to measure cortisol binding globulin (CBG) in serum. It is a microtitre plate, solid phase, reagent excess, sandwich assay in which the same polyclonal antiserum is used as a source of capture and labeled antibodies. The results of this assay were shown to be reliable and were fully comparable with those obtained by a commercially available kit. As a reflection of the free cortisol concentration we measured cortisol and CBG concentrations in serum and calculated the Free Cortisol Index (FCI) = [cortisol]serum/[CBG]serum.100. The clinical use of this parameter, as a screening test for disturbances of the pituitary-adrenal axis, was investigated in different groups of subjects: healthy men and women, women using oral contraceptives, pregnant women at term, patients with thyroidal illnesses, patients using anti-epileptic drugs and patients suffering from adrenal diseases. In a number of groups we compared the FCI results with measurements of cortisol in saliva, another parameter used as an estimate of the concentration of free cortisol in blood. Our conclusion is that the FCI, in contrast to a total cortisol measurement alone, can prevent unnecessary further testing.

A time-resolved immunofluorometric assay of autoantibodies to double-stranded DNA.

A time-resolved immunofluorometric assay (TRIFMA) of autoantibodies to double-stranded DNA (dsDNA) is described. Biotinylated DNA was bound to the polystyrene solid phase coated with streptavidin. F(ab1')2 fragments from antibodies raised in rabbits to human IgG were labelled with Eu3+, and used in the assay to label the bound autoantibodies to dsDNA. The measuring range covers three decades. The proposed assay has good analytical properties. For calibration the First International Standard for antibodies to double-stranded DNA Wo/80 was used. The 97.5th percentile in normal persons is 20 kIU/l. Comparison of the TRIFMA and the Farr-assay in the analysis of 56 sera from 20 patients with systemic lupus erythematosus or lupus-like disease, 13 with other autoimmune diseases, and 10 blood-bank donors indicates a high degree of concordance (Kendall's tau = 0.56, p < 0.001). Further evaluation in 102 patients with systemic lupus erythematosus, lupus-like disease and other autoimmune diseases shows that sensitivity for active classical systemic lupus erythematosus is adequate, and specificity is excellent.

Temperature effects on free-thyroxine measurements: analytical and clinical consequences.

We compared the analytical and clinical performance of two free-thyroxine (FT4) assays--a solid-phase radioimmunoassay, Spectria, and a time-resolved fluoroimmunoassay, Delfia, both of them two-step methods--with the performance of a direct radioimmunoassay, Nichols, to measure FT4 concentration in equilibrium dialysate of undiluted serum. The three assays showed comparable analytical performance. We tested clinical utility in sera from 135 healthy subjects with and without thyroxine-binding abnormalities and in 61 patients with and without thyroidal illnesses. We found significant differences for FT4 measured by different assays in sera from the same euthyroid patients. To explain the differences, we studied the influence of temperature on performance and calibration. Most important was the neglected fact that the association constant for the binding of thyroxine to thyroxine-binding globulin decreases when the temperature rises from 20 to 37 degrees C, causing a doubling of FT4. The two-step assays, if performed at room temperature without a well-defined calibration, can give misleading FT4 concentrations. This is the case when sera from patients with thyroxine-binding abnormalities are measured against kit standards, made up in normal human sera. If an assay is to reflect the in vivo FT4 concentration at body temperature in all types of samples, it should be performed at body temperature. For practical reasons 37 degrees C is recommended, and reference values should be defined at 37 degrees C. The same might be valid for other free-hormone assays.

A time-resolved fluoroimmuno assay of the IgM-rheumatoid factor.

A time-resolved fluoroimmuno assay of the IgM-rheumatoid factor is described. Aggregated rabbit IgG was coated to microtitre plates to serve as the target protein. F(ab')2-fragments from antibodies, raised in rabbits against human IgM, were labelled with Eu3+ and used in the assay to mark the bound IgM-rheumatoid factor. The labelling procedure is easy to perform, and there is no need for special equipment. The shelf life of the label at -20 degrees C is at least one year. The lower detection limit of the assay is 1.3 x 10(3) IU/l. The range over which the IgM-rheumatoid factor can be measured at a within-run precision of less than 5% without varying the dilution (working range) is 5-1200 x 10(3) IU/l. Linearity in serum dilutions is good. There is good correlation with existing methods for the assay of IgM-rheumatoid factor. This correlation is better with an assay using rabbit IgG as the target than with one using human IgG. Comparison of methods shows that standardization, despite the use of the WHO Reference Preparation as the first calibrator, remains problematic. The 95th percentile in normal bloodbank donors is 8 x 10(3) IU/l. The costs for the reagents were about 0.5 Dutch florin (ca 0.30 US-$) per well. In conclusion, the method described here is analytically at least comparable with other methods, in precision, linearity, working range etc. Finally, it is easy to perform.

Common variable immunodeficiency in a family.

Common variable immunodeficiency (CVID) is mainly characterised by hypo- or agammaglobulinaemia of late onset, usually discovered in the second decade of life. In individuals CVID is associated with a variable impairment of cellular immunity and the susceptibility to microbial infection may vary as well. CVID is described in a mother and a son who suffered from serious bacterial infections. In addition, minor immunological test abnormalities of apparently healthy first degree relatives are described.

Evaluation and modification of a radioimmunoassay for pregnancy-specific beta 1-glycoprotein.

The radioimmunoassay available from Behringwerke for pregnancy-specific beta 1-glycoprotein (SP 1) was tested for its ability to detect pregnancy prior to the first missed menstrual period. It was found that the equine serum, used as solvent for the standards, did not react like human serum. The standard solvent was replaced by bovine serum albumin 50 g/1 and pooled human serum respectively. Equilibrium and sequential incubation procedures were compared. The latter appeared to be more sensitive in the low value range and was therefore more suitable for the early detection of pregnancy. Also, with standards in albumin, the sequential assay was more specific. SP1 could be detected in sera of men and non-pregnant women, using albumin as standard solvent. This could be due to different cross reacting material of the protein matrix, or to the presence of SP1- like material in human sera. The choice of human male serum seemed to be the most practical. It has also been adopted by Behring, and a commercial kit has been prepared.

[Reference values for lactate dehydrogenase in the serum during childhood and puberty (author's transl)]. / Referenzwerte für die Lactatdehydrogenase im Serum im Kindesalter und in der Pubertät.

Lactate dehydrogenase activity was measured in the serum of persons between the ages of 1 and 22 years. There was a continuous decrease with age, which occurred in three phases. The most striking was the marked decrease at the age of about 12 years. The normal adult range is reached at about 22 years.

Optimized radioimmunoassay curve fitting program suitable for use on desktop calculators.

A simplex search program for the logit-log parameters of radioimmunoassay calibration curves, suitable for use on a small desktop calculator, is described. The technique features a weighted least-square method to extract the maximum information out of the calibration data. A criterion for rejection of outliers is included.