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The regulations for quality management in the rheumatology laboratory are demanding and on a qualitatively high level. They are the result of continuously more detailed quality requirements and their implementation. Not only the process of correctly measuring analytes but also aspects of preanalytics, postanalytics and interpretation of the results as well as validation of new test methods are becoming increasingly more important for the diagnostic process.

Kinetic properties of the Na-Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca(2+) concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca-Ca exchange mode, a transient inward current is observed, whereas in the Na-Ca exchange mode, current either rises to a plateau, or at higher Ca(2+) concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca(2+) concentrations jumps activate Na-Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca(2+) of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 muM). Measurement of the Ca(2+) dependence of the rising phase provides direct evidence that the association rate constant for Ca(2+) is about 5 x 10(8) M(-1) s(-1) and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca(2+)-dependent rate constant, which is about 9,000 s(-1) at saturating Ca(2+) concentrations. The voltage independence of this relaxation is maintained for Ca(2+) concentrations far below saturation. In the Ca-Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge-voltage relation is independent of the Ca(2+) concentration. Major conclusions are: (1) Photolytic Ca(2+) concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca(2+) at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca(2+) at the cytoplasmic transport sites is high (5 x 10(-8) M(-1)s(-1)) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca(2+) translocation at saturating Ca(2+) concentrations is valid for Ca(2+) concentrations far below saturation.

This third part of this series of articles on laboratory diagnostics of rheumatic diseases considers the rheumatic diseases caused by infection by microorganisms, or reactive arthritides. The basis for laboratory diagnostics of infection-reactive arthritides is the investigation of anti-infection antibodies. In some situations, DNA amplification methods may be helpful. Bacterially infected joints should be immediately examined by arthrocentesis and microscopic examination and laboratory culture of the synovial fluid.

This is the second part in a series of articles on the laboratory diagnostics of rheumatic diseases. It addresses rheumatoid arthritis, systemic, anti-neutrophil cytoplasmatic antibody (ANCA) positive vasculitides and antiphospholipid-syndrome. The diagnostics of rheumatoid arthritis has been substantially improved by the recently introduced assay for antibodies against citrullinated peptides. In addition, a number of vasculitides can be differentiated by the presence of ANCA. Beta2-glycoprotein I antibodies and lupus anticoagulants are at present the most specific markers for antiphospholipid syndrome. Inflammatory activity can be monitored by determining the levels of acute phase proteins and erythrocyte sedimentation rate, but only in some situations by measuring immunoglobulins and interleukins.

This is the first part of a series of articles on the laboratory diagnostics of rheumatic diseases and will consider the systemic autoimmune diseases lupus erythematosus, Sjögren's syndrome, systemic sclerosis, dermato/polymyositis and mixed connective tissue disease (MCTD, SHARP syndrome). The basis for diagnostics is the presence of antinuclear antibodies (ANA). Initially, these antibodies are detected using a screening test. This must be followed by the identification of the patient's individual autoantibody specificities, which then yields important diagnostic clues. Disease activity may be monitored serologically by following the titers of selected autoantibodies and, in certain patients, by examining complement consumption.

The activation kinetics of the endogenous Ca(2+)-activated Cl(-) current (I (Cl,Ca)) from Xenopus oocytes was investigated in excised "giant" membrane patches with voltage and Ca(2+) concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca(2+) concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca(2+)-dependent. The lag phase decreases from about 10 ms after a small Ca(2+) concentration jump (0.1 microM) to less than 1 ms after a saturating concentration jump (55 microM). The rate constant of the rising phase is half-maximal at about 5 microM. At saturating Ca(2+) concentrations, the rate constant is 400 to 500 s(-1). The Ca(2+) dependence of the stationary current can be described by the Hill equation with n=2.3 and K (0.5)=0.5 microM. The amplitude of the stationary current decreases after the excision of the membrane patch with t (1/2) approximately 5 min (run-down). The activation kinetics of the current elicited by a Ca(2+) concentration jump is not affected by the run-down phenomenon. At low Ca(2+) concentration (0.3 microM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca(2+) levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca(2+)-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.

The induction of lupus-like syndromes with the appearance of single-stranded DNA antibodies is a well-known complication of drug therapy. In this report we present a patient with an erosive seropositive rheumatoid arthritis developing the clinical and serological features of systemic lupus erythematosus including the occurrence of double-stranded DNA antibodies under sulfasalazine treatment.

In order to investigate, if complement levels can be used as an indicator of clinical activity in systemic lupus erythematosus (SLE), levels of C3, C4, CH50, and C3d were measured in 79 patients, 41 with inactive, 31 with moderately active and 7 with severely active disease. Our study shows that C3d, and particularly the C3d/C3 ratio, provide sensitive markers for disease activity in SLE. Since C3d is a direct measurement of complement turnover, it reflects complement activation better than C3, C4 and CH50.

The effect of low external Na+ concentrations on the light-induced K+ release from crayfish photoreceptor cells was tested by labelling intracellular K+ with the isotope 86Rb. The amount of isotope released per light stimulus is roughly proportional to the external Na+ concentration if the osmolarity is kept constant by replacing Na+ with Tris, choline or sucrose. When sucrose is used to replace the depleted Na+ the light-induced K+ release is a linear function of the external Na+ concentration and is reduced by approx. 95% at an external Na+ concentration of 5 mmol/l. For choline and Tris substitutions the relationships are less clear but at Na+ concentrations less than or equal to 56 mmol/l it seems that in comparison with sucrose the light-induced K+ release is smaller in a Tris solution and larger in a choline solution. It is suggested that the light-induced K+ release is due mainly to an activation of voltage sensitive K+ channels.

1. Membrane current responses produced by intracellular ionophoretic injection of Ca2+ ions into voltage clamped Helix neurones were investigated. 2. The Ca2+ activated current depends on the intensity and duration of the Ca2+ injection as well as on the membrane potential. The current-voltage relation is non-linear and shows strong outward rectification. 3. The Ca2+ activated current reverses near the equilibrium potential for K+ ions and the associated current fluctuations disappear at the K+ reversal potential, which indicates a single current source, probably activation of K+ channels. 4. The Ca2+ activated K+ conductance is voltage dependent and changes e-fold per 31 mV membrane depolarization. 5. Extracellular tetraethylammonium, gallamine and quinidine block the Ca2+ activated K4 current. Extracellular 4-aminopyridine has no blocking effect. 6. Sustained injection of high amounts of Ca2+ irreversibly suppresses the outward current. 7. The K+ outward current evoked by repeated Ca2+ injection exhibits summation and facilitation.

In voltage-clamped "motor-giant" neurones of the crayfish Orconectes limosus a depolarizing voltage step elicits a transient inward current carried by Na+ which is followed by an early and a delayed outward current. The early outward current is reduced if the Na+ current is suppressed by tetrodotoxin or the removal of external Na+. It is also abolished if the K+ channel blocking agents tetraethylammonium and 3,4-diaminopyridine are applied to the neurone. The outward current was not depressed if Li+ was substituted for Na+ in the external solution or if the Na-K pump was inhibited by ouabain or the removal of external K+. Ionophoretic injections of EGTA did not depress the early outward current. Short ionophoretic injections of Na+ into the neurone increased the outward current elicited by a depolarization but did not affect the leakage current. It is suggested that the influx of Na+ leads to a transient increment of the Na+ concentration near K+ channels and that internal Na+ ions exert an activating or modulating effect on K+ channels.

The effects of three toxins (ATX I, II, III) isolated from the sea anemone Anemonia sulcata were studied in the soma membrane of a crustacean neurone under voltage-clamp conditions. All three toxins affected the action potentials and the Na+ currents in a similar manner. The lowest concentrations tested (10 nM, 20 nM and 50 nM for ATX I, II and III, respectively) had pronounced selective effects on the Na+ current. No effect on K+ or Ca2+ currents was observed with concentrations up to 5 microM. In the presence of ATX the Na+ inactivation was incomplete even with pulses of 700 ms length or strong depolarizing prepulses. Besides the effects on the inactivation process ATX affected also the activation of the Na+ current. In cells treated with ATX the negative resistance branch of the peak Na+ current voltage relation was shifted by -5 mV to -20 mV. The time to peak was increased for small depolarizations (up to -30 mV) and the rate of rise (delta I/delta t) was enlarged by ATX. A slow activating current component was also observed after depolarizing prepulses or if the Na+ current was outward. The decay of the Na+ tail currents was considerably prolonged after the application of ATX if the membrane was repolarized to potentials more positive than about -60 mV. Repetitive stimulation led to a shortening of the action potential in ATX II treated neurones. A simultaneous and parallel decrement of the peak and plateau current was observed with depolarizing voltage steps.