Comparative study of C reactive protein and serum amyloid A protein in experimental inflammation.

The responses of C reactive protein, measured by radial immunodiffusion and radioimmunoassay, and serum amyloid A protein, measured by radial immunodiffusion, were compared in eight subjects with inflammation induced experimentally by intradermal injection of monosodium urate crystals. A significant increase in serum amyloid A was noted after a lag phase of eight hours, the increase in median concentration at 48 hours being about eightfold. A parallel but less marked increase was found in C reactive protein when measured by radioimmunoassay (fourfold increase in median concentration at 48 hours) after a small but significant decrease during the lag phase. The changes in C reactive protein remained within the reference range and were not detectable by radial immunodiffusion.

External quality assessment of total urinary protein estimation in the United Kingdom.

Three surveys of total urinary protein quantitation have been carried out in 350 UK laboratories. The seven specimens comprised buffered saline or normal human urine with added human serum albumin or human serum, or urine from individuals with nephrotic syndrome. Principal method groups were: turbidimetry (57%), dye binding (25%) and biuret (15%). For all surveys, overall between-laboratory agreement was poor (CV 22.8% to 57.1%), with ranges of results from 24-fold (0.83-20 g/L) to 366-fold (0.05-18.3 g/L); there was no improvement with time. The most popular method (sulphosalicylic acid turbidimetry) consistently performed the worst, and performance of the direct biuret procedure was also unacceptable; both methods should be discontinued. There were no significant differences in performance between the other major method groups, and none can be specifically recommended. Within the individual calibrant groups, least variation was observed with human serum. A common calibrant for all participants yielded significantly better between-laboratory agreement for all methods except sulphosalicylic acid turbidimetry.

Alpha 1-microglobulin, beta 2-microglobulin and retinol binding protein in childhood febrile illness and renal disease.

Serum and urinary levels of alpha-1-microglobulin (A1M), beta-2-microglobulin (B2M) and retinol binding protein (RBP) were measured using a Mancini radial immunodiffusion technique in 52 children with renal disease, 36 with non-renal febrile illness and 29 controls. In controls the mean serum level for A1M was 25 +/- 4.6 (SD) mg/l for B2M 1.7 +/- 0.5 mg/l and for RBP 31 +/- 8 mg/l. A1M levels were not significantly altered by febrile illness while B2M was elevated and RBP markedly depressed. Serum A1M and B2M were elevated in the nephrotic syndrome, while serum B2M was also raised during infancy. Coefficients of log-transformed data with creatinine-derived glomerular filtration rate (GFR) were -0.87 for B2M, -0.71 for RBP, and -0.62 for A1M. In the urine A1M was always measurable in controls while B2M and RBP were undetectable in all but a small number. The urine levels of all three proteins increased in response to non-renal febrile illness, and rose invariably when GFR fell to below 40-50 ml/min per 1.73 m2. Of the three proteins A1M was most frequently elevated in the urine with febrile and renal illness. RBP was rarely detectable when the other two proteins were not. Urinary A1M was consistently elevated in the nephrotic syndrome in contrast to B2M, possibly as a reflection of the increased glomerular permeability. We conclude that serum B2M is superior to A1M and RBP as an index of glomerular filtration, although its levels should be interpreted with caution in febrile disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical assays of serum proteins: a European external quality assessment survey and the effects of calibration procedures on interlaboratory agreement.

An external quality assessment survey of immunochemical assays of 9 proteins (immunoglobulins G, A and M, complement components C3 and C4, alpha1-antitrypsin, orosomucoid, haptoglobin and transferrin) in 5 European countries (Austria, France, Hungary, Italy and UK) showed inter-country differences in the mean values obtained. Reprocessing of the results using one of the two specimens distributed as a 'calibrant' effectively eliminated or reduced substantially these differences. Consideration of the methods used by participants confirmed previous indications from national surveys that the differences were due to lack of agreement among commercial calibrants. Such interlaboratory variations were also minimised by the 'calibration' in this survey. The role of European working calibration materials in ensuring interlaboratory agreement on an international basis is discussed.

Anionic salivary proteins associated with connective tissue disorders: sialated tissue kallikreins.

Parotid saliva was collected from 32 patients with rheumatoid arthritis, 10 with systemic lupus erythematosus, three with mixed connective tissue disease, 12 with progressive systemic sclerosis, two with primary Sjögren's syndrome, and four with Raynaud's syndrome. Tissue kallikreins were measured by radioimmunoassay, and saliva samples were subjected to isoelectric focusing followed by immunoblotting or silver staining. The results showed that the saliva of patients with connective tissue diseases contained increased amounts of immunoreactive tissue kallikrein. In addition, there was an increase in the multiple forms of anionic tissue kallikreins, resulting mainly from a shift in their distribution towards that of higher sialic acid content and lower isoelectric point. These changes were most obvious in patients with systemic lupus erythematosus. Novel or unusual glycosylation may explain the occurrence of increased amounts of anionic salivary proteins in connective tissue diseases.

Stability of alpha 1-microglobulin, beta 2-microglobulin and retinol binding protein in urine.

The stability of alpha 1-microglobulin (alpha 1M), beta 2-microglobulin (beta 2M) and retinol binding protein (RBP) in urine was determined in 135 random samples from children with renal disease, febrile illness, malignancy, and from controls. Immediately after voiding, samples were divided into two portions, one of which was alkalinized. After identical transit times and laboratory handling the pH and concentrations of the individual proteins in each pair were measured. beta 2M was unstable in urine of pH less than 7 and grossly so below pH 6. In some instances beta 2M was low or undetectable even in the alkalinized samples when alpha 1M and RBP levels were raised, suggesting that degradation of beta 2M may have occurred prior to voiding. Concentrations of alpha 1M and RBP were significantly lower in the non-alkalinized fractions at pH less than 7, although to lesser degree than for beta 2M. Contrary to previous reports, we conclude that the stability of all 3 proteins is affected by urinary pH and recommend that this be measured and alkalinisation performed at the time of voiding.

Urinary total protein estimation--fact or fiction?

In two recent surveys of urinary total protein assays, 370 laboratories in the United Kingdom were requested to determine the protein content of a simulated 24-hour urine (a solution of sodium and potassium salts and urea, with no added protein) and of a 24-hour urine from a healthy individual. The nature of these specimens was not revealed and participants used their routine methods and calibrants. Quantitative results (range 0.005-12.23 g/l, median 0.03 g/l) were received from 31% of the participants for the salt solution and from 43% for the normal urine (range 0.01-2.96 g/l, median 0.05 g/l). Nonquantitative results, i.e. those given as less than a detection limit (range less than 0.005 to less than 0.5 g/l) were received from 29% of the participants for the salt solution and from 33% for the normal urine. Statements of 'nil', 'zero' or 'not detected' were received from the remainder. Further analysis of the results indicated that 29% of the laboratories reported, or did not unequivocally exclude, significant proteinuria in the salt solution, and 41% of the laboratories similarly did not exclude proteinuria in the normal urine. It is proposed, for both clinical and analytical reasons, that consideration be given to the discontinuation of urinary total protein estimation and that urinary albumin, supplemented where appropriate by other selected protein or enzyme measurements, be determined instead.

Serum amyloid A protein compared with C-reactive protein, alpha 1-antichymotrypsin and alpha 1-acid glycoprotein as a monitor of inflammatory bowel disease.

The serum concentrations of serum amyloid A protein (SAA), C-reactive protein (CRP), alpha 1-antichymotrypsin (alpha 1-ACT) and alpha 1-acid glycoprotein (alpha 1-AGP) have been measured in eighty-six patients with Crohn's disease, twenty-five patients with ulcerative colitis and twenty-two patients with the irritable bowel syndrome. In the Crohn's and ulcerative colitis group significant increases in concentration were observed in all four proteins, which parallelled disease severity as defined by other conventional laboratory parameters formulated into a simple activity index. In the irritable bowel group no significant changes were seen. Serum amyloid A and CRP concentrations were significantly lower in ulcerative colitis than in Crohn's disease when mild, but did not differ significantly when severe. Serum amyloid A correlated well with CRP (r = 0.83) and alpha 1-ACT (r = 0.80), but less well with alpha 1-AGP (r = 0.65). Serum amyloid A was the most sensitive protein (77%) but had the lowest specificity (74%). C-reactive protein was less sensitive (58%) than SAA but had greater specificity (100%). Alpha 1-ACT had a sensitivity and specificity similar to CRP and, therefore, provided little or no additional information. Alpha 1-AGP, although also 100% specific, had the lowest sensitivity (34%) and, therefore, is probably the least useful acute phase monitor of inflammatory bowel disease. The role, and associated problems, of SAA measurements are discussed.

Overestimation of immunoglobulins in the presence of rheumatoid factor by kinetic immunonephelometry and rapid immunoturbidimetry.

The United Kingdom External Quality Assessment Scheme for Specific Proteins has demonstrated that one method group, comprised of kinetic immunonephelometric assays, gives significantly higher results for immunoglobulins in certain pathological sera containing rheumatoid factor. Using IgM as a model, we have compared kinetic immunonephelometry and a rapid (5 min incubation) immunoturbidimetric assay with radial immunodiffusion (RID) on seven sera. Compared with RID significantly higher results were obtained by kinetic nephelometry on all five sera positive for rheumatoid factor, and by turbidimetry on three of the four of these studied. In a serum with IgM increased due to primary biliary cirrhosis lower results (P0.005) were obtained by nephelometry than by RID. The remaining serum was normal, and no significant intermethod differences were found. The possible causes for these discrepancies are discussed.

Improved between-laboratory agreement for specific protein assays in serum following introduction of a common reference preparation (SPS-01) demonstrated in an external quality assessment scheme.

Between-laboratory agreement for 6 specific protein assays was better in two surveys (total 8 specimens) in which a calibration material was provided than in the preceding five surveys (20 specimens) in a national external quality assessment scheme. CVs for immunoglobulins G, A and M (200 participants) and alpha 1-antitrypsin and complement components C3 and C4 (70 participants) were reduced from 18.3% (range 11.6-23.8%) to 10.7% (range 7.5-13.6%). This improvement was not due to changes in within-laboratory precision or in the concentrations of protein surveyed. Improvement was maintained in the following four surveys (3 normal and 5 pathological sera) without calibration material for immunoglobulins G and A and for alpha 1-antitrypsin. Agreement for immunoglobulin M, C3 and C4 returned to values close to the original CVs, but subsequently improved. The role of a common calibration material in improving between-laboratory agreement is discussed.

Systemic response to local urate crystal induced inflammation in man: a possible model to study the acute phase response.

The production of a systemic inflammatory response to intradermal monosodium urate crystal injection is described. A transient, self-limiting local response is associated with a systemic response detectable by a rise in the white cell count and serum amyloid A protein. The white cell change parallels the evolution of the local response, whereas the serum amyloid A response lags behind the local lesion, peaking after the local lesion is resolving. Intradermal monosodium urate injection is proposed as a possible inflammatory stimulus to explore the acute phase protein response in different disease states.

Acute phase response of serum amyloid A protein and C reactive protein to the common cold and influenza.

C reactive protein (CRP) and serum amyloid A protein (SAA) are sensitive and rapid acute phase reactants, and their measurement for monitoring inflammatory disease and assessing the prognosis in secondary amyloidosis is gaining widespread acceptance. The changes in these proteins in eight subjects suffering from natural colds, 15 subjects with experimentally induced colds (rhinoviruses E1, 3, 9, 14, or 31), and eight with experimentally induced influenza (A/Eng/40/83) were studied. SAA concentration increased in 21 of the 23 subjects with natural or experimental rhinovirus colds (mean increase 95 mg/l); CRP concentration increased in 11 (mean increase 11 mg/l). All subjects with influenza showed pronounced increases in SAA concentrations (mean increase 642 mg/l) while six showed increases in CRP concentration (mean increase 22 mg/l). All these increases were highly significant (p less than 0.001). Asymptomatic excretors of both rhinovirus and influenza virus showed significant increases in SAA concentration (p = 0.015 for rhinovirus and p less than 0.001 for influenza virus) but not in CRP concentration. No changes in SAA or CRP values were seen in 12 volunteers after challenge with saline. These observations suggest that caution is required in the interpretation of estimations of SAA concentration and that it may be too sensitive an acute phase protein for clinical use as its concentration may be raised in both trivial and asymptomatic viral infections.

External quality assessment of immunoassays for specific proteins in serum; 18 months' experience in the United Kingdom.

Five surveys of immunoglobulin A, G and M assay have been carried out in 200 UK laboratories; assays of complement components C3 and C4 and alpha 1-antitrypsin (alpha 1-AT) by 70 laboratories were also included in the fourth and fifth surveys. Clarified liquid human serum was used for specimens, two pairs of specimens related by dilution being used in each survey. For immunoglobulins, no inter-method bias was noted although one calibrant gave significantly different mean values; nephelometry, as used by one method group (largely comprising Beckman ICS users), and radial immunodiffusion gave the best inter-laboratory agreement for IgA and IgM, while IgG showed little method dependence. Inter-laboratory precision was better for C4 than for C3 and alpha 1-AT, which indicates the importance of calibrant differences for the latter two; there were too few participants to allow reliable conclusions about methods to be drawn.

Serum amyloid-A protein concentration in rheumatoid arthritis and its role in monitoring disease activity.

The serum concentrations of serum amyloid-A protein (SAA), C-reactive protein (CRP), and alpha 1-acid glycoprotein (alpha 1-AGP) have been measured in 185 patients with rheumatoid arthritis. SAA and CRP concentrations correlated well (r = 0.86) both within and above the normal ranges, though SAA showed a greater incremental increase than CRP. All patients with normal SAA levels also had normal CRP and alpha 1-AGP concentrations. In contrast, in 40% of patients with normal CRP and alpha 1-AGP concentrations the SAA was raised, sometimes markedly so. The clinical and serological assessments of disease activity in these patients were not significantly different from those with concomitantly raised levels of CRP. These findings suggest that SAA is a more sensitive marker of inflammation than is CRP. The role of the measurement of SAA as a monitor for inflammatory disease activity is discussed.

Quantitative radial immunodiffusion assay for serum amyloid A protein.

A radial immunodiffusion assay for serum amyloid A protein (SAA) using a commercially available antiserum is described. Serum is applied untreated to 1% agarose gels prepared in 0.02 M barbitone buffer, pH 8.6, containing 40 g/l polyethylene glycol 6000. Incubation is carried out overnight at 37 degrees C. The assay combines the advantages of simplicity, rapidity, specificity and stability, and avoids the hazards associated with the previously described radioimmunoassays. The method has sufficient sensitivity to measure SAA in the majority (99%) of normal subjects, and confirms the behaviour of SAA as a very sensitive acute phase reactant in inflammatory disease. The method is ideally suited to the rapid processing of a large number of samples.