This prospective study evaluated the diagnostic utility of measuring calprotectin in ascites to identify ascitic PMN counts > 250/μL in patients referred for paracentesis, and provides the following new information: Patients with an elevated PMN count (> 250/μL) had higher ascitic calprotectin levels than those with normal cell counts; this finding indicates that ascitic calprotectin levels correlate well and reliably with PMN count. It is clinically significant that calprotectin levels in ascitic patients can identify elevated PMN counts using both laboratory-based ELISA and bedside POC testing. Indeed, ascitic calprotectin may serve as a surrogate marker for PMN count and would be amenable to routine SBP screening, especially when measured by a bedside test.
Ascites is commonly found in patients with liver cirrhosis and may promote bacterial translocation, enhancing the risk of SBP[3
]. SBP in outpatients is rare, but when it occurs it often requires hospitalisation to manage to disease course[4,5
]. In our study, four of 71 patients were diagnosed with SBP (5.6%). In general, SBP symptoms are nonspecific and current guidelines recommend paracentesis be performed in all patients with ascites to rule out abdominal infection[6,7
]. The diagnosis of SBP in patients with liver cirrhosis is based on a PMN count of > 250/μL in ascitic fluid, with or without positive bacterial cultures[5-7
]. This cut-off is recognized as more sensitive than other criteria (PMN > 500/μL; white blood cell count > 500/μL)[38,39
] for identifying SBP[40
]. SBP diagnosis based solely on bacterial culture is considered unreliable, since up to 60% of patients with increased PMN count are reported as culture-negative[41,42
]. In our study, all patients with culture-positive abdominal infection, including both SBP and secondary peritonitis patients, had elevated PMN counts. In the four SBP patients, the bacterial cultures were positive for only one (25.0%).
Our study measured calprotectin in ascitic fluid in 130 unselected samples from 71 consecutive patients. Ascitic calprotectin levels correlated well and reliably with PMN counts, and the samples with PMN > 250/μL also had higher ascitic calprotectin levels than the samples with PMN ≤ 250/μL. Both the ELISA and the POC test accurately measured ascitic calprotectin, and the correlation between the two tests was excellent with high sensitivity (95% and 100%, respectively) and high specificity (89% and 85%, respectively) at the optimal cut-off points (from ROC analysis). In a diagnostic test that is used to screen for a specific disease, it is preferred to test all patients at risk, especially when potentially life-threatening complications may occur. In screening tests, high sensitivity is therefore favoured over high specificity. In our study, the NPVs of calprotectin testing in ascites were excellent (99% for the ELISA and 100% for the POC test). Notably, these results suggest that no patient with elevated PMN count would have been missed by the bedside test.
In daily clinical practice, PMN count is often not readily available and clinicians frequently rely on total cell count when initiating empiric antibiotic treatment[43
]. It has been suggested that a total cell count < 1000/μL (obtained from automated cell counting procedures) is unlikely to signify SBP, having a NPV of 96%[44
]. In our study, using such a criteria would have misclassified five patients (26.3%) with elevated PMN counts. Moreover, the use of total cell count in combination with ascitic calprotectin measurement did not increase the diagnostic accuracy of calprotectin testing, as calculated by ROC analysis (data not shown).
To avoid diagnostic delay, it has been proposed that automated PMN counting should replace the laborious and time-consuming manual cell counting technique[8,9
]. Studies have demonstrated that automated blood cell counts correlate well with manual ascitic leukocyte differential counts[45
]. However, despite the potential benefit of automated cell counters in clinical practice, widespread use of this technology is limited by the cost of the sophisticated laboratory equipment and requirement for trained operators; this is a particular challenge for practitioners’ office settings and small clinics without in-house laboratories.
The use of reagent strips (urine dipsticks) for PNM counting (by colorimetric detection of leukocyte esterase activity) has also been evaluated as a rapid SBP diagnostic tool[45,46
]. A number of these studies have reported sensitivities between 85% and 100% and specificities between 90% and 100%[10-25
]. However, these results came from mostly single-centre studies with small numbers of SBP cases. The only large, multicentre study reported in the literature produced very different results; in particular, using 2123 paracenteses, the sensitivity was only 45% for identifying PMN > 250/μL in cirrhotic patients[26
]. Although specificity was still high, it was concluded that urinary dipstick testing lacks sufficient accuracy for diagnosing SBP. The risk of false negative results seemed to be especially problematic in patients with lower PMN counts[46
]. These results dampened the initial enthusiasm for reagent strips, and currently this method is not recommended for rapid diagnosis of SBP[45
Only one study in the literature, to date, has provided data on calprotectin measurement in ascites[35
]. In that study, Homann et al[34
] compared ascites from patients with malignant disease to ascites from patients with non-malignant disease. Higher ascitic calprotectin levels were found in the malignant patients and shown to correlate with increased mortality in patients with decompensated liver cirrhosis. However, the authors did not investigate the diagnostic potential of ascitic calprotectin. More recently, Parsi et al[27
] measured ascitic lactoferrin (an iron-binding protein also found in PMNs) in cirrhotic patients with ascites and investigated its potential for identifying SBP. The lactoferrin measurements correctly identified PMN counts > 250/μL in 22 of 218 samples (10.1%), yielding a sensitivity of 95.5% and specificity of 97.0%. However, the quantitative assay (ELISA) used in that study is not commercially available, and to date no bedside test, qualitative or quantitative, exists for lactoferrin.
The results from our current study confirm the findings reported by Parsi et al[27
]. Specifically, we show that measurement of calprotectin, a leukocyte-specific protein, may serve as a surrogate marker for the PMN count in ascitic fluid. A particular strength of our study is the quantitative measurement of calprotectin by two methods, a laboratory-based ELISA and a commercially available bedside test. Rapid bedside measurement is advantageous for hospitalised patients, as it supports early antibiotic intervention and limits unnecessary treatments. It will also benefit the outpatient setting by providing on-site testing, since samples are otherwise required to be transported to an offsite laboratory. The POC test that we used can accomplish quantitative measurement of ascitic calprotectin within minutes, and this feature is expected to minimize the problems associated with diagnostic delay that clinicians currently face. Additionally, the cost of POC testing may be less than the other methods, such as contracting with the offsite laboratories.
There are several limitations to the current study that merit consideration. First, the prevalence of SBP in our study cohort was lower than expected from the literature. Second, we included all patients with ascites, irrespective of the aetiology, and it may be that our results cannot be generalised to all patients with liver cirrhosis. Third, this was an exploratory study that aimed to establish the concept of measuring a PNM-related inflammatory protein, rather than PNM cells themselves, as an indicator of elevated cell count in ascites; therefore, no formal sample size calculation was performed. Finally, our sample size was small and larger studies are needed to evaluate this test in different clinical settings and to establish a reliable cut-off for ascitic calprotectin for optimal identification of PMN counts > 250/μL.
In conclusion, we have demonstrated that measurement of calprotectin in ascitic fluid correlates well with the PMN count and reliably predicts levels > 250/μL. Additionally, we showed that an elevated PMN count could easily be measured by a POC test device which may enable a treating physician to obtain useful bedside measurements, especially those practicing in settings with limited equipment and/or technical personnel. Further studies are warranted to define a clinically useful cut-off for the diagnosis of SBP in cirrhotic patients with ascites.