Els Dumoulin

Proteolysis is a confounding factor in the interpretation of faecal calprotectin

Abstract Background: Calprotectin is a 36 kDa calcium and zinc binding protein. An increased level of calprotectin points towards inflammatory bowel disease. However, the biomarker calprotectin shows 14 potential cleavages sites for trypsin. Next to trypsin, also the presence of its inhibitor α1-antitrypsin after a gastrointestinal bleeding may affect calprotectin testing. In this study, effects of trypsin and α1-antitrypsin as potential confounders for faecal calprotectin testing are investigated. Methods: An in vitro model was created. As calprotectin source, leukocytes were isolated and subsequently lysed (1% Triton X-100) and diluted in faecal matrix. Trypsin digestion was carried out by adding trypsin. Incubation occurred for 24 h or 48 h (37 °C). To study the influence of α1-antitrypsin on trypsin, the same experiment was repeated after adding serum containing α1-antitrypsin. Results: In vitro experiments enabled monitoring of the faecal calprotectin digestion, leading to loss of immunoreactivity. Trypsin activity was a potential confounder in the interpretation of calprotectin, in particular for proximal lesions, where exposure of calprotectin to trypsin is prolonged. Relative calprotectin loss was proportional to the amount of trypsin. Decrease of calprotectin was more pronounced after 48 h of incubation in comparison to 24 h of incubation. Analogue experiments also showed stable calprotectin values after adding α1-antitrypsin. Conclusions: Transit time, trypsin activity and addition of blood as a source of α1-antitrypsin may be regarded as potential confounders in the interpretation of calprotectin results. Age-related cut-off values depending on the anatomical localisation of the lesions could improve the diagnostic efficiency of calprotectin testing.

Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease

Abstract Background: Leukocyte cytosolic proteins (e.g., calprotectin) are emerging biomarkers for inflammatory bowel disease. Leukocyte aryl esterase activity has been commonly used for sensitive detection of leukocytes in human body fluids such as urine. Urine test strip results are generally reported in categories. As automated strip readers allow quantitative data to be reported, sensitive quantitative detection of leukocytes in body fluids has become possible. Here, we explored the use of leukocyte esterase as a potential alternative faecal biomarker for inflammatory bowel disease. Methods: We evaluated leukocyte esterase activity in faecal extracts and compared Cobas u 411 (Roche) quantitative reflectance data with calprotectin concentration for 107 routine samples. Stability of leukocyte esterase for trypsin digestion was carried out by adding trypsin to the extract. Incubation occurred at 37 °C for 24 h or 48 h. Results: Reproducibility of the reflectance signal was good (within-run imprecision: 6.1%; between-run imprecision: 6.2%). Results were linear in the range 103–106 WBC/100 mg faeces. The lower limit of detection was 4 WBC/μL and the lower limit of quantification was 5 WBC/μL. Stability of LE activity in stool and faecal matrix was good. An adequate correlation was obtained between leukocyte esterase activity and the faecal calprotectin concentration: log(y)=4.28+0.29log(x). In vitro experiments monitored the digestion of leukocyte esterase and faecal calprotectin. Leukocyte esterase activity was significantly less affected by trypsin activity than calprotectin immunoreactivity. Conclusions: Quantitative leukocyte esterase activity of faecal extracts provides information about the leukocyte count in the gut lumen. Leukocyte esterase is a promising and affordable alternative biomarker for monitoring inflammatory bowel disease.

Investigation of sensitivity for coagulation factor deficiency in APTT and PT: how to perform it?

The activated partial thromboplastin time (APTT) has been used for years for different purposes including screening for intrinsic factor deficiencies (FVIII, FIX, FXI and FXII) [1, 2]. The APTT factor sensitivity depends on reagent composition comprising the type of contact activator as well as the origin and composition of phospholipids [3, 4]. Current guidelines recommend a prolongation of the APTT when factor levels are below 30% [5]. However, mild deficiencies above 30% can also imply a significant risk of bleeding [6, 7]. Prothrombin time (PT) is primarily used to monitor oral vitamin K antagonists (VKA). Again, different reagents are available with different sensitivities [8]. Literature is very scarce but Testa et al. demonstrated a discrepant sensitivity to FV, FVII, and FX of two PT re agents [8]. Different sources of thromboplastins (rabbit, human, recombinant) may influence the FVII dosage [9]. Equally, a prolongation of the PT when factor levels drop from 30% to 45% is recommended [5]. By the use of dilution series of normal frozen plasma and freeze-dried or frozen factor deficient plasmas, the responsiveness of different APTT reagents has been tested [4]. Lawrie et al. suggested that lyophilized deficient plasmas may contain procoagulant material [10]. They concluded that determination of APTT factor sensitivity in accordance with the CLSI guidelines can give inconsistent and misleading results. The use of well-defined patient samples was recommended [10]. Reagent composition and laboratory equipment are the most important variables for observed differences in sensitivity. Every laboratory should assess their own factor sensitivities in order to correctly interpret their routine coagulation tests. In this study, we aim to evaluate and compare the factor sensitivities in our daily practice by two appraoches: dilution experiments with normal pooled plasma (NPP) and in accordance with the CLSI guidelines by the use of patient deficient plasmas. APTT (STA-PTTA, Diagnostica Stago, Asnières, France) and PT (Neoplastine CI Plus, Stago) were performed on the STA-R Evolution (Stago). One-stage-clotting assays were performed for intrinsic factors with STA-C.K. Prest (Stago) and for extrinsic factors with Neoplastine CI Plus (Stago), and by the use of lyophilized STAImmunodeficient (STA-ID) (Stago) (FII, FVIII, FIX, FXI and FXII) and lyophilized STA-deficient (STA-D) (Stago) (FV, FVII, and FX) plasmas. In-house made NPP consisting of equal volumes of 80 healthy volunteers and stored at –80 °C was used for dilutions. Factor levels of NPP, STA-ID and STA-D plasmas were measured. Factor sensitivity of APTT (FVIII, FIX, FXI and FXI) and PT (FII, FV, FVII and FX) was assessed by two experiments. First, a spiking experiment was performed by mixing increasing volumes of NPP with factor deficient plasma. A series of 10 samples with factor activities of 10% up to 100% was obtained. Second, an analog series of ±22 patient samples was selected for every factor (two different patients per level). If certain factor activities lacked, different samples were mixed. A limited number of patients with FX deficiency and no patients with FII deficiency were available. APTT (s) or PT (s) and the studied factor (%) were analyzed in duplicate whereas other intrinsic or extrinsic factors were analyzed in single. If factor levels were outside the reference range besides the studied factor, samples were excluded. A difference of 5% for duplicates was accepted [5]. The factor sensitivity is defined as the level of factor from where on a prolongation of APTT or PT was observed. Factor sensitivities were

Bing-Neel syndrome: Two unexpected cases and a review of the literature.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by the proliferation of small B-lymphocytes in the bone marrow that produce monoclonal immunoglobulin M (IgM). We describe two patients with WM who presented with neurological symptoms due to infiltration of lymphoplasmacytoid tumor cells in the central nervous system, a condition known as Bing-Neel syndrome. A literature review revealed that this syndrome is rare and commonly missed in clinical practice due to its variable presentation and a lack of awareness or knowledge. Brain and spinal magnetic resonance imaging may show a focal mass or diffuse infiltration. The diagnosis of Bing-Neel syndrome requires proof of IgM or lymphoplasmacytoid cells in cerebrospinal fluid or in a brain biopsy. Treatment with intravenous and/or intrathecal chemotherapy and cranial radiotherapy is described in literature with generally poor outcome, although a combination of these therapies seems to improve outcome. Nevertheless, insufficient data are currently available to make general treatment recommendations.

Dual-wavelength recording, a simple algorithm to eliminate interferences due to UV-absorbing substances in capillary electrophoresis.

Analytical interferences have been described due to the presence of various exogenous UV‐absorbing substances in serum. Iodine‐based X‐ray contrast agents and various antibiotics have been reported to interfere with interpretation of serum protein pherograms, resulting in false diagnosis of paraproteinemia. In the present study, we have explored the possibility of measuring UV absorbance at two distinct wavelengths (210 and 246 nm) to distinguish between true and false paraproteins on a Helena V8 clinical electrophoresis instrument. This study demonstrates that most substances potentially interfering with serum protein electrophoresis show UV‐absorption spectra that are distinct from those of serum proteins. Scanning at 246 nm allows detection of all described interfering agents. Comparing pherograms recorded at both wavelengths (210 and 246 nm) enables to distinguish paraproteins from UV‐absorbing substances. In case of a true paraprotein, the peak with an electrophoretic mobility in the gamma‐region decreases, whereas the X‐ray contrast media and antibiotics show an increased absorption when compared to the basic setting (210 nm). The finding of iodine‐containing contrast media interfering with serum protein electrophoresis is not uncommon. In a clinical series, interference induced by contrast media was reported in 54 cases (of 13 237 analyses), corresponding with a prevalence of 0.4%. In the same series, 1631 true paraproteins (12.3%) were detected. Implementation of the proposed algorithm may significantly improve the interpretation of routine electrophoresis results. However, attention should still be paid to possible interference due to presence of atypical proteins fractions (e.g., tumor markers, C3).

Determination of iohexol and iothalamate in serum and urine by capillary electrophoresis

Estimation of glomerular filtration rate (eGFR) is essential to assess kidney function. Iodine‐containing contrast agents detection by HPLC has been proposed as a safe alternative for inulin or radioactive compounds. However, HPLC is a time‐consuming and labor‐intensive method. The aim of this study was to develop an assay for iohexol and iothalamate using capillary electrophoresis. Iohexol and iothalamate were directly analyzed by CE in serum and urine, using photometric detection (246 nm). Serum peak height was proportional to iohexol and iothalamate concentrations. Detection limits for iohexol and iothalamate were 10 and 5 mg/L. Limits of quantification were 13.0 and 15.0 mg/L. Within‐run CVs were 4.9 and 6.5%; between‐run CVs 3.1–9.9% and 3.8–13.7%. A good correlation was observed between CE and HPLC: y = 1.1703x + 5.017 (iohexol) and y = 0.7807x + 11.01 (iothalamate; (y = concentration obtained by CE [mg/L], x = concentration obtained by HPLC [mg/L]). In addition, CE allowed to determine urinary iohexol concentration. Although the detection limit for CE was higher than for HPLC, CE can still be used for eGFR determination. Advantages of this high‐throughput method are the absence of sample pretreatment and a minimal sample volume requirement.

Trypsin is a Potential Confounder in Calprotectin Results.

3. T o the Editor: Dhaliwal et al (1) related intestinal inflammation and growth in children with cystic fibrosis (CF), reporting that most pancreatic-insufficient patients with CF showed increased levels of fecal calprotectin, whereas other markers (S100A12 and osteoprotegerin) of inflammation were not elevated. The pancreatic-sufficient group with CF had normal fecal calprotectin levels. Dhaliwal et al hypothesized that this finding was because of predominant non-neutrophilic gut inflammation in patients with CF; however, in this study, inflammation was not confirmed by endoscopy and histology. Although intestinal inflammation in patients with CF has been reported (2), the underlying pathological mechanism remains unknown (3–5). Increased fecal calprotectin in pancreatic-insufficient patients with CF, however, cannot exclusively be attributed to intestinal inflammation because fecal calprotectin is subject to proteolysis by trypsin. Intraluminal trypsin activity markedly declines from proximal to distal. Because bowel length in children is shorter, exposure time to proteolysis by trypsin is shortened, making fecal calprotectin less susceptible to trypsin proteolysis. Recent findings, based on an in vitro model, demonstrate that endogenous trypsin may be a confounder in the interpretation of fecal calprotectin data (6). These findings are in agreement with earlier reports on calprotectin and proximal inflammation (7,8). Endogenous trypsin activity in pancreatic insufficient patients with CF is virtually absent. As a result, calprotectin proteolysis is also reduced. Therefore, calprotectin levels detected in stools of pancreatic-insufficient patients may be relatively higher (6). The effects of pancreatic enzyme replacement therapy on stool calprotectin have not been reported to date. In conclusion, intestinal trypsin levels may confound the results of fecal calprotectin in pancreatic-insufficient patients with CF.