Ivan Brandslund

Double-decker rocket immunoelectrophoresis for direct quantitation of complement C3 split products with C3d specificities in plasma

A double-decker rocket immunoelectrophoresis (DD-RIE) method for direct quantitation of complement split products with C3d determinants in human plasma is described. The usefulness of the DD-RIE method for monitoring C3 activation has been assessed and compared with conventional crossed immunoelectrophoresis (CIE) for C3c determination in a patient with iatrogenic septic shock and patients with rheumatoid arthritis. In contrast with CIE the DD-RIE method is quantitative by reference to a standard curve based on an internal reference C3d preparation and its sensitivity and assay capacity are superior to CIE. All reagents and antibody preparation are commercially available and the production of standards is easy. No overlapping was observed between C3d values in plasma from healthy persons and patients with active classical rheumatoid arthritis. The DD-RIE is highly suitable for routine use in laboratories of clinical immunology.

Optimization of preanalytical conditions and analysis of plasma glucose. 1. Impact of the new WHO and ADA recommendations on diagnosis of diabetes mellitus

The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucosein blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1h until centrifugation at minimum 1000 g for 10min. The plasma is then stable for at least 48h at room temperature.The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8 mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucose in blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1 h until centrifugation at minimum 1000 x g for 10 min. The plasma is then stable for at least 48 h at room temperature.

Can capillary whole blood glucose and venous plasma glucose measurements be used interchangeably in diagnosis of diabetes mellitus

According to new proposals from the American Diabetes Association (ADA) and WHO, venous peripheral plasma is the preferred system for measuring glucose for diagnosing diabetes mellitus. Owing to the instability of glucose in plasma after blood sampling, strict well-defined and standardized preanalytical conditions are essential to ensure that glucose concentration measured in plasma reflects real blood glucose in the patient. This is in contrast to the capillary whole blood measurements, which are easy to perform and well established. We investigated whether it is possible to perform analysis on capillary whole blood but express the results as plasma glucose values and hence obtain comparable results and the same predictive values for diagnosis in the individual patient? The conclusion of our investigations is that these two systems are not interchangeable and that conversion should not be done for diagnostic purposes where plasma determinations are recommended.

Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll®)

A standardized, reproducible two-step method for separation of human peripheral blood monocytes on continuous Percoll gradients has been developed. The first step involves separation of mononuclear cell on Percoll of density 1.075 g/ml and the second step separation of monocytes from lymphocytes on a continuous Percoll gradient with a starting density of 1.075 g/ml for the formation of the gradient. The average yield during a 10 month period of daily routine use has been 74 +/- 17% (mean +/- 1 S.D.), and the average purity 63 +/- 10%. Ninety to 95% of the monocytes are viable after separation as judged from trypan blue exclusion and by ingestion of latex particles and sensitized sheep erythrocytes. The separation takes about 3 h and the total number of monocytes obtained from 40 ml of blood is in the range of 10-15 x 106. The procedure has been reliable with 3-4% separation failures, mainly due to bacterial or fungal growth in Percoll suspension or media. The contaminating cells are exclusively lymphocytes, predominantly T-lymphocytes (90-95%), when citrate is used as anticoagulant. Heparin can not be used as anticoagulant, as there appears to be a dose-dependent formation of thrombocyte aggregates which contaminate the monocytes, and result in poor separation.

A standardized method for quantitating the complement-mediated immune complex solubilizing capacity of human serum.

A standardized radioassay for measuring the complement-mediated immune complex solubilizing capacity (CMSC) and the initial kinetics of the solubilization (IKS) reaction is described. The total complement (C)-mediated solubilizing capacity was determined after incubation of diluted serum and 125I-BSA-anti-BSA. Percentage C-mediated solubilization (CMS) was measured after centrifugation by determining the distribution of radioactivity. The dependency of CMSC upon factors such as serum dilution and buffer system used, amount of IC added to serum, serum storage conditions and centrifugation conditions was investigated in order to optimize the assay. The CVt of the standardized assay was 0.10-0.17 depending upon the CMSC level measured. Treatment which inactivates C factors (heating), interferes with C activation (EDTA) or activates and consumes C components (zymosan) markedly reduces the CMSC. Preliminary investigation of pathological sera showed that both IKS and CMSC were clearly reduced in SLE sera. By contrast, rheumatoid arthritis sera exhibited normal IKS and only marginal reduction in CMSC.

Circadian and Diurnal Variation of Circulating Immune Complexes, Complement-mediated Solubilization, and the Complement Split Product C3d in Rheumatoid Arthritis

Nine patients with active classical rheumatoid arthritis (ARA criteria) were studied with reference to circadian variation of immunological and clinical parameters. Complement-mediated solubilization (CMS) of immune complexes (IC) and the level of circulating IC were found to be inversely related with low CMS and increased IC levels in the morning, and vice versa in the afternoon. Bed rest and exercise did not influence these fluctuations. The C3d concentration in plasma was increased but showed no diurnal or circadian periodic fluctuations when the levels were corrected for fluctuations in plasma albumin concentration. Clinical assessment by means of pain score exhibited marked variations, with high scores in the morning, and lower in the daytime, whereas measurements of Ritchies joint index showed no consistent pattern. The circadian variations in CMS, serum IC and clinical parameters indicate the need to collect blood specimens and perform clinical examinations of patients at a fixed time of day.

STEROIDS AND COMPLEMENT ACTIVATION IN RHEUMATOID ARTHRITIS

: Patients with seropositive, classical rheumatoid arthritis (RA) with severe active disease have raised plasma concentrations of the complement C3 split product C3d. These values display little diurnal or circadian variation in the individual patient. During a 3-month period the variation was within 10 mU/l in 45 patients (ref. range 20-52 mU/l, RA patients up to 120 mU/l.) Six RA patients were treated with steroids on clinical indication, and the plasma C3d, Ritchie index and pain score before and during the treatment (30 mg prednisolone per day) were measured. The variables showed a steady decrease during the next 14 days. Plasma C3d fell 2/3 of the total fall within the first 48 hours, while the serum total haemolytic complement activity, complement C3 and C4 did not change significantly. This shows that the anti-inflammatory effect of steroids is accompanied by a reduction of complement activation.

Production of antibody against C3c epitopes which are inaccessible on native C3

Anti-C3c antiserum was induced by immunizing rabbits with autologous erythrocytes coated with human C3b/C3bi. By absorption with human plasma this antiserum was rendered specific for C3c epitopes which are not expressed on native C3. This specificity may be analogous to that of immunoconglutinin.

Simultaneous quantitation of free haptoglobin and haemoglobin-haptoglobin (HbHp) complexes by double-decker rocket immunoelectrophoresis

Existing immunochemical methods for haptoglobin (Hp) determination are incapable of distinguishing between free haptoglobin and haemoglobin-haptoglobin (HbHp) complexes. A one-dimensional double-decker rocket immunoelectrophoretic method (DD-RIE) for simultaneous quantitation of free Hp and Hp bound as HbHp complexes was therefore developed. The DD-RIE can be used for quantitative in vitro studies of the Hb-Hp interactions, for quantitation of Hps Hb binding capacity and for investigations of haemolytic episodes in patients. The clinical value of the method is illustrated by an investigation of a patient with a gradual accumulation of HbHp complexes in plasma, a steady rise in total Hp and a fall in free Hp during haemolysis. A proposed receptor-mediated clearance of the complexes by the reticuloendothelial system (RES) could not be substantiated, as HbHp receptors were not demonstrable on human blood monocytes.

Catabolism of haemoglobin-haptoglobin complexes in haemolytic uraemia-like syndromes of different etiologies

The catabolism of haemoglobin-haptoglobin complexes was studied in four patients with increased vascular haemolysis as part of acute or subacute haemolytic uraemic syndromes. The apparent volumic substance elimination rates for haemoglobin (Fe) bound to haptoglobin in plasma were 1.1 mumol/h/l and 2.9 mumol/h/l in two patients suffering from sublimate and hydrochloric acid poisoning, respectively. This is estimated to correspond to a normal catabolism, when the increased haptoglobin synthesis is taken into account. In the other two patients suffering from serum-sickness there was reduced clearance and thereby an accumulation of haemoglobin-haptoglobin complexes in plasma during penicillin administration. When the offending drug was withdrawn the plasma concentration of haemoglobin bound to haptoglobin remained high for about three days and then fell rapidly (approximately with 3.8 mumol/l/h and 1.9 mumol/l/h). Thus, also in these patients the clearance capacity could be normalized after discontinuation of the drug.