Exploratory study on reference intervals of calprotectin and pentraxin 3

Introduction

Reference intervals (RIs) play a key role in the process of evaluating laboratory test results. The most commonly used RI range is the 95% interval, which includes 95% of the central laboratory test values found in the reference population, that is, healthy individuals. Accurate and reliable RIs are important tools for clinicians in the medical decision-making process and adequate patient management. In addition, high clinical standards maintained by healthcare professionals, in addition to the use of precise RIs and evidence-based clinical guidelines, require the implementation of novel diagnostic tools for effective and precise disease activity assessment. Specifically, over the years, scientists have been intensively searching for novel markers that are early and specific indicators of either systemic or localized inflammation processes. Among many potential biomarkers, calprotectin and pentraxin 3 (PTX3) have emerged as promising markers of various pathophysiological conditions, including autoimmune diseases, infections, cardiovascular diseases, and even cancer.^1–5

Calprotectin is a dimeric complex of two calcium-binding subunits, S100A8 and S100A9, which are located in the cytoplasm of neutrophils, monocytes, and macrophages and are released from cells upon their activation. ⁶ Its use as a stool biomarker has been found to be as a sensitive and specific laboratory tool for assessing intestinal inflammation and differentiating between inflammatory bowel disease (IBD) and functional gut disorders.^7,8 In addition to calprotectin, pentraxin 3 is an inflammatory protein secreted from a variety of cell types (neutrophils, macrophages, endothelial and epithelial cells, myeloid cells, dendritic cells, etc.) in response to cytokine stimuli and/or endotoxins/bacterial products, and thus takes part in the regulation of the inflammatory response.^9–11 Although PTX3 has shown promising results in various diseases, unlike calprotectin, PTX3 has not yet been used in a clinical setting.

In addition, both calprotectin and pentraxin 3 have been widely investigated as potential inflammation markers in different body fluids, including serum, plasma, urine, synovial fluid, and ascites. Of those mentioned, serum/plasma has shown significant analytical and preanalytical variability of calprotectin measurement caused by either tube type, storage conditions, or different manufacturer kits, which consequently reflects the reference interval ranges.^12–15 Although the preanalytical variability of pentraxin 3 in serum/plasma has not been investigated thoroughly, manufacturer data suggest differences in measurement depending on tube type. ¹⁶ Unlike serum/plasma, even though calprotectin has been investigated as a potential marker for acute/chronic bladder or kidney conditions, to the best of our knowledge, no study has established reference intervals for calprotectin in urine samples.

Taken together, our study aimed to determine reference intervals for serum pentraxin 3 and calprotectin, as well as urine calprotectin via a commercially available turbidimetric method – the Bühhlmann fCAL® turbo Reagent Kit and the Pentraxin 3 commercially available ELISA kit

Materials and methods

The study was conducted at University Hospital Sveti Duh from December 2023 until March 2024, according to the Clinical and Laboratory Standard Institute (CLSI) EP28-A3C guidelines for defining, establishing, and verifying reference intervals in the clinical laboratory. ¹⁷ The study was conducted in accordance with the Declaration of Helsinki and its protocol was approved by the hospital’s ethical committee.

Results

The studied populations included a group of 60 male and 60 female participants whose serum calprotectin and pentraxin 3 levels were measured. The population had a median age of 43 years (19–77). The average CRP concentration of the studied population was 1.5 ± 0.9 mg/L, while the leucocyte count was 6.4 ± 1.4 × 10⁹/L. The calprotectin reference range was found to be ≤3.6 mg/L, with the 90% CI for the upper reference range being 3.1–4.1 mg/L, whereas the RIs for PTX 3 was found to be ≤3.0 µg/L, with the 90% CI for the upper reference range being 2.7–3.2 µg/L. The histogram results for the studied population for both serum calprotectin and pentraxin 3 are presented in Figure 1.

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Figure 1.

Relative frequency (%) distributions of the concentrations of calprotectin (a) and pentraxin 3 (b) in serum.

The demographic data of the participants included in the urinary calprotectin RIs are presented in Table 1. The urinary calprotectin concentration was found to be ≤ 1.4 mg/L, with a 90% CI for the upper reference range of 1.0–1.7 mg/L. The histogram results for the studied population are presented in Figure 2.

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Table 1.

Demographic data of the population included in the urinary calprotectin determination.

Demographic parameter	Value
N (total number of participants)	120
Age (years)	54 (24–75)
Leukocytes (x109/L)	6.2 (5.3–7.0)
Serum creatinine (µmol/L)	73 (63–83)
Serum urea (mmol/L)	5.2 (4.4–6.1)
eGFR (ml/min/1.73 m2)	92 (83–101)

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Figure 2.

Relative frequency (%) distributions of calprotectin concentration in the urine samples.

Discussion

Our study has three major findings. First, previous studies have investigated the role of calprotectin in various bladder and kidney conditions, however, this is the first report of urinary calprotectin reference ranges established according to laboratory standard guidelines that can assist in distinguishing between healthy and pathological conditions. Second, this is the first report of calprotectin reference intervals in serum samples using the immunoturbidimetric Bühhlmann fCAL® turbo Reagent Kit applied on an automated chemistry analyzer. Finally, our study is the first to investigate and report pentraxin 3 reference intervals in serum samples collected from a Caucasian population.

Urinary calprotectin has emerged as a promising marker in chronic kidney disease, bowel/kidney inflammatory conditions, and cancer.^20,21 In these studies, its concentration was measured mostly with commercially available ELISA kits on a different set of control populations. For example, Sahin and colleagues enrolled 52 patients and 30 control subjects in their study where they investigated whether urinary calprotectin could be used as a biomarker in the diagnosis of primary bladder cancer and as a diagnostic tool in determining high-grade and stage disease. ²⁰ The control subjects enrolled in the study were male participants who underwent transurethral resection of the prostate. The median urinary calprotectin concentration measured by the R&D ELISA kit was 32.96 ng/mL (IQR 14.39–410.04 ng/mL). Similarly, in a study performed by Seibert and coworkers enrolled 29 healthy subjects without any medical history. ²¹ They measured calprotectin from urine samples stored at −20°C (storage length not defined) via a commercially available ELISA kit and reported a median calprotectin concentration of 56.9 µg/L (IQR 13.3–124.6 µg/L). Both of these studies measured somewhat lower calprotectin concentrations than we found in our study. These variations could be attributed to differences in measurement methodologies (immunoturbidimetry vs. ELISA), the number of enrolled subjects (120 in our study vs. 30 and 29 in the mentioned studies), population age (median 54 years vs. 68 and 74 years), sex or the fact that calprotectin was measured in fresh or previously frozen samples, etc. However, the strength of our study lies in the fact that we conducted it in accordance with the CLSI EP28-A3C guidelines in fresh urine samples from outpatients who had all blood, serum, and urine laboratory results within the RIs and no medical history records of any acute/chronic inflammatory diseases/conditions or acute/chronic kidney/urinary bladder diseases or cancer.

Circulating calprotectin and pentraxin 3 concentrations have been proposed as promising biomarkers for monitoring disease activity in patients with rheumatoid arthritis and Takayasu’s arteritis, respectively.^22,23 However, their shortcoming as potential biomarkers is substantial preanalytical and analytical variability depending on the sample type, centrifugation, storage conditions, and analytical assay used.^12,15,24,25 Many studies have previously investigated the levels of calprotectin and pentraxin 3 in healthy individuals in either serum or plasma samples.^{12,19,26–38} The number of included individuals ranged from as low as 10 to 166. The mean calprotectin concentrations ranged from 0.1 mg/L to 2.5 mg/L in the serum samples, whereas the median PTX3 concentration ranged from 0.1 to 3.9 ng/mL in the plasma samples. Such differences could have been attributed to the number of recruited healthy participants or preanalytical conditions such as precentrifugation time or even storage conditions (i.e. −20, −80, or −140°C). As with the latest reported results, it remains unclear whether the differences found for calprotectin might be attributed to the analytical procedure used since Navejan and colleagues reported that reference values were comparable across four assays for serum and lithium heparin samples – EliA™ Calprotectin 2 assay on Phadia™200 (Thermo Fisher Scientific; serum/plasma protocol research use only), MPR8/14 ELISA (Bühlmann Laboratories; CE marked, research use only USA), QUANTA Flash® Circulating Calprotectin CIA on BIO-FLASH® (Werfen; CE marked, research use only USA) and Diasorin Calprotectin assay on Liaison (Diasorin S.P.A; serum/plasma protocol research use only). ²⁴ Nevertheless, considering the wide range of calprotectin reference values in healthy populations found by different research groups, it seems that analytical assays might influence both calprotectin and pentraxin 3 measurement results, or that either the preanalytical conditions or the term ‘healthy population’ needs to be specified in more detail when reporting study results. In our study, we report for the first-time reference values for calprotectin using a commercially available Bühlmann PETIA assay applied on an automated analyser and pentraxin 3 for BioVendor ELISA for healthy individuals with CRP and leukocytes within the reference range, and no medical records of acute/chronic inflammatory diseases/conditions or cancer were included. Additionally, the samples were centrifuged within a maximum of 2 h of sampling, and all the samples were analysed within 6 h of blood collection, with which we tried to reduce the preanalytical conditions to a minimum. To conclude, before further larger studies on specific preanalytical and analytical conditions, it is advisable to apply local population and method-based reference intervals for both calprotectin and pentraxin 3.