Dialysis patients have a large burden of cardiovascular disease, with both traditional Framingham and many ‘non-traditional’ risk factors identified. One such non-traditional risk factor is inflammation. A syndrome of malnutrition, inflammation and atherosclerosis has been identified in dialysis patients and implicated in the pathogenesis of cachexia and cardiovascular disease [9
]. Pathologically, inflammation is a known contributor to intimal atherosclerotic disease in which macrophages and other inflammatory cells are thought to be critical in the pathogenesis of plaques [12
]. Inflammation has been shown to lead to reduced fetuin-A levels in serum, likely making patients more vulnerable to extraskeletal calcification when this serum calcium-binding protein is decreased [13
]. The advent of the high-sensitivity CRP has led to a plethora of studies in the general population and in dialysis patients – nearly all showing a strong association of elevated levels with cardiovascular and all-cause mortality. However, inflammation is an end event of numerous inciting factors and mediators, and many of the biomarkers utilized to define or diagnose inflammation are part of the acute phase response, which is a non-specific diffuse response initiated by multiple factors [14
In the present study, we documented that these biomarkers change with acute events such as infection and stroke, with recovery as the event recovers. Thus the accuracy of the biomarkers for acute inflammation is not questioned. However, we found large variances in patients who were clinically stable, and this variance was not explained by having a TC as opposed to an AVF. TC have been hypothesized to contribute to intermittent inflammation and elevated biomarkers of inflammation [8
]. In the present study, patients with TC were more likely to get infected, but in patients without clinically apparent infections, the presence of TC did not explain the variance. Between 30 and 46% of the total variation was due to within-patient variation despite assay performance (CV) that was within manufacturer recommendations.
Similar estimates of variance have been previously reported in a smaller study in dialysis patients. A study of 29 patients, excluding subjects with temporary catheters, measured levels weekly for 16 months and found intrasubject variance even higher, with 71.3 and 86.7% of total variation for CRP and IL-6, respectively [15
]. An equally poor variance was found for serum amyloid A protein, another inflammatory biomarker [15
]. In contrast, estimates of variation in the general population are much lower. In one study, the intraindividual variation for ln(CRP) for healthy adults was 14% compared to 9% for those with angina [16
]. Thus, this variance is substantially higher in dialysis patients than in the healthy adults, even in those with native fistulas. The reason for the variation could not be assessed from this study as we did not perform random blood cultures, dental examinations, tagged white cell scans or other searches for clinically hidden causes of inflammation.
Unfortunately, the lack of stability of these biomarkers makes practical use problematic. Clinical use of the biomarkers to identify patients at risk or who have smoldering infections is not likely valid given that, for example, over one-third of subjects changed from one tertile of CRP to another in the course of just 3 months. This is consistent with the study of van Tellingen et al. [17
], who found patients frequently changed tertiles of CRP and concluded that clinical judgment was as predictive of infection as was a rise in CRP levels. The intrapatient variation will need to be taken into account if any of these biomarkers are used as a surrogate endpoint in research studies that employ specific interventions aimed at reducing inflammation. Certainly, sample size calculations for studies using these biomarkers in dialysis patients should take into account this variation and study designs should include repeated measurements to be certain that the observed effect is stable over time. Our data support the need to validate that a change in these biomarkers prospectively predicts a change in clinical endpoints prior to their use as a valid surrogate endpoint. Lastly, authors should report their own laboratories' CV when using these assays. Nearly all samples analyzed in the present study required dilution of the sample to remain within the manufacturer's standard curve, which may create increased assay error.
There are several limitations to our study. First, this is a relatively small sample size although the largest reported to date. We only followed patients for 4 months, and perhaps longer follow-up, or more frequent follow-up would have altered our findings, although Tsirpanlis et al. [15
], measuring weekly values found similar variability. We also only measured four potential inflammatory biomarkers, all of which have limited specificity for inflammation. Lastly, there may have been some selection bias in those patients enrolled. Our patients were predominately African-American, had a high percentage of glomerulonephritis and were younger than the average US dialysis population. However, these apparently clinically stable patients may be enrolled in clinical trials and thus the results provide important information on the limitations of using these biomarkers in clinical trials.
In summary, there is considerable intrapatient variation in biomarkers CRP, IL-6, fetuin-A and albumin. Whether this phenomenon is a problem with the assays, a problem with our patients or both is a question that remains to be answered. Given problems with circulating fragments in other peptides such as PTH [18
], interference in assay measurements by inactive fragment or other uremic toxins is a possibility. However, a more likely explanation for the high and fluctuating levels in dialysis patients is that these biomarkers can be elevated by a number of processes, and thus may simply reflect the multiple co-morbid illnesses present in our patients. Unfortunately, this means further studies are needed to refine the precision of these biomarkers before they can be considered true surrogate markers for cardiovascular disease in dialysis patients. Until that time, clinical studies of anti-inflammatory measures should evaluate clinically meaningful endpoints to prove efficacy.