Measuring calprotectin in plasma and blood with a fully automated turbidimetric assay

Calprotectin in plasma and blood might prove to be a useful biomarker of inflammation and infection; however, automated methods for analysing the concentration of calprotectin in those materials are lacking. We have validated a fully automated turbidimetric method and present health-related reference limits. Calprotectin was measured by Siemens Advia XPT with the Bühlmann fCAL® turbo test (Bühlmann Laboratories AG, Schönenbuch, Switzerland), a particle enhanced turbidimetric immunoassay for quantification of calprotectin in fecal extracts. Plasma and serum samples were analysed directly, while whole blood was first extracted with M-PER® Mammalian Protein Extraction Reagent (ThermoFisher) and diluted with B-CAL-EX (Bühlmann). We studied analytical imprecision, estimated health-related reference limits and examined the correlation between neutrophil-calprotectin (blood-calprotectin adjusted for plasma-calprotectin) and the neutrophil count. The intermediate (‘day-to-day’) coefficient of variation was 3.5 and 1.0% for heparin-plasma-calprotectin at 0.52?mg/L and 3.53?mg/L, respectively, and 4.9% for heparin-blood-calprotectin at 50.2?mg/L. Health-related reference limits were 0.470–3.02?mg/L for calprotectin in heparin-plasma, 50.8–182?mg/L for calprotectin in heparin-blood, 0.534–2.41% for the ratio between them and 24.7–33.3?pg for the mean amount of calprotectin per neutrophil. Compared to heparin-plasma, calprotectin concentrations were significantly lower in EDTA-plasma and higher in serum (p?<?.05). Correlation between neutrophil-calprotectin and the neutrophil count was excellent. We have shown that the Bühlmann fCAL® turbo test can be used to measure calprotectin in plasma and blood.     

Age-specific seroprevalence of poliomyelitis,diphtheria and tetanus antibodies in Spain

In 1996, a seroepidemiological study was undertaken in Spain, with the main aim of estimating the population's immunity against poliomyelitis, tetanus and diphtheria. A population-based cross-sectional study was conducted, covering the population aged 2-39 years. The sample was stratified by age and rural-urban environment, and informed consent obtained to take blood specimens from subjects attending phlebotomy centres. The study included 3,932 persons and the prevalence of antibodies against all three types of poliovirus exceeded 94% across all age groups. From a high of 96% in subjects under the age of 15 years, immunity against diphtheria steadily declined to a low of 32.3% in subjects aged 30-39 years. Similarly, tetanus antitoxin concentrations indicating basic protection were present in 98.9% of the under-14 years age group; thereafter, immunity declined, until reaching 54.6% in the 30-39 years age group.     

Novel pathogenic LRRK2 p.Asn1437His substitution in familial Parkinson's disease

Genealogical investigation of a large Norwegian family (F04) with autosomal dominant parkinsonism has identified 18 affected family members over four generations. Genetic studies have revealed a novel pathogenic LRRK2 mutation c.4309 A>C (p.Asn1437His) that co‐segregates with disease manifestation (LOD = 3.15, θ = 0). Affected carriers have an early age at onset (48 ± 7.7 SD years) and are clinically asymmetric and levodopa responsive. The variant was absent in 623 Norwegian control subjects. Further screening of patients from the same population identified one additional affected carrier (1 of 692) with familial parkinsonism who shares the same haplotype. The mutation is located within the Roc domain of the protein and enhances GTP‐binding and kinase activity, further implicating these activities as the mechanisms that underlie LRRK2‐linked parkinsonism. © 2010 Movement Disorder Society     

MRI in evaluation of perianal fistulae

Background

Fistula is considered to be any abnormal passage which connects two epithelial surfaces. Parks’ fistulae classification demonstrates the biggest practical significance and divides fistulae into: intersphincteric, transsphincteric, suprasphincteric and extrasphincteric. Etiology of perianal fistulae is most commonly linked with the inflammation of anal glands in Crohn’s disease, tuberculosis, pelvic infections, pelvic malignant tumours, and with the radiotherapy. Diagnostic method options are: RTG fistulography, CT fistulography and magnetic resonance imaging (MRI) of pelvic organs.

Patients and methods

We have included 24 patients with perirectal fistulae in the prospective study. X-rays fistulography, CT fistulography, and then MRI of the pelvic cavity have been performed on all patients. Accuracy of each procedure in regards to the patients and the etiologic cause have been statistically determined.

Results

29.16% of transphincteric fistulae have been found, followed by 25% of intersphincteric, 25% of recto-vaginal, 12.5% of extrasphincteric, and 8.33% of suprasphincteric. Abscess collections have been found in 16.6% patients. The most frequent etiologic cause of perianal fistulae was Crohn’s disease in 37.5%, where the accuracy of classification of MRI was 100%, CT was 11% and X-rays 0%. Ulcerous colitis was the second cause, with 20.9% where the accuracy of MRI was 100%, while CT was 80% and X-rays was 0%. All other etiologic causes of fistulae were found in 41.6% patients.

Conclusions

MRI is a reliable diagnostic modality in the classification of perirectal fistulae and can be an excellent diagnostic guide for successful surgical interventions with the aim to reduce the number of recurrences. Its advantage is that fistulae and abscess are visible without the need to apply any contrast medium.     

A dose-escalating phase I of imatinib mesylate with fixed dose of metronomic cyclophosphamide in targeted olid tumours

Background:

Preclinical findings suggest that imatinib mesylate (IM) and metronomic cyclophosphamide (MC) combination provides synergistic antiangiogenic activity on both pericytes and endothelial cells.

Methods:

We have designed a 3+3 dose-escalating phase I trial with a fixed dose of MC (50 mg two times daily) plus IM (400 mg per day; 300 and 400 mg two times daily). Enrolled patients had IM- and sutininib-refractory advanced gastrointestinal stromal tumours (GIST) (n=17), chordoma (n=7) and mucosal melanoma (n=2). Dose-limiting toxicities were monitored for the first 6 weeks. Progression-free survival (PFS) and response assessment are based on RECIST 1.0 guidelines. Pharmacokinetics of IM were measured before and after exposure to MC.

Results:

No dose-limiting toxicity was observed. Fourteen patients of the expanded cohort received 400 mg two times daily of IM with MC. Apart from a case of possibly related acute leukaemia occurring after 4 years of treatment, we did not see unexpected toxicity. No drug–drug pharmacokinetic interaction was observed. There was no objective response. We have observed long-lasting stable disease in chordoma patients (median PFS=10.2 months; range, 4.2–18+) and short-term stable disease in heavily GIST pretreated patients (median PFS=2.3 months; range, 2.1–6.6).

Conclusion:

This combination is feasible and may warrant further exploration in refractory GIST or chordoma patients.     

Injecting drug use as risk factor for visceral leishmaniasis in AIDS patients

In Madrid Region 6652 AIDS cases were diagnosed between 1982 and 1993. Visceral leishmaniasis (VL) was present in 166 (2.49%). VL frequency among injecting drug users proved higher than that for the other transmission categories (relative risk 2.57; 95% confidence interval 1.64–4.01). This could point to an alternative Leishmania transmission route via needle sharing.     

Trends in hepatitis A virus infection with reference to the process of urbanization in the greater Madrid area (Spain)

Hepatitis A is an infection transmitted by the fecal-oral route. Endemicity within a specific country is directly related to sanitation and hygienic standards, while being inversely related to socioeconomic conditions. We studied how the process of urbanization witnessed in Madrid had influenced the transmission of hepatitis A infection. In the Madrid Autonomous Region, this process first began in the early sixties and was not brought to a close until the late seventies. Catalytic models were used to estimate the annual infection rate, , on the basis of seroprevalence data stratified by age. A cohort effect related to a fall-off in infancy-related hepatitis A virus (HAV) is to be observed in the results for the last few years. The model permits four birth cohort-based groups to be differentiated by : individuals born pre-1960, =0.082 (95% CI 0.095–0.070); those born in the early sixties, =0.052 (95% CI 0.060–0.042); whose members were born in the late sixties, =0.033 (95% CI 0.041–0.025); and those born in the late seventies, =0.017 (95% CI 0.020–0.013). The first group includes those born before the urbanization process had started. The second and third groups coincide with the development stage of that process, hence exhibiting transitional rates. The fourth group reflects the process in its consolidation stage. This reduction in the transmission of infection has changed the manner of presentation, so that while isolated cases or small outbreaks tend to be more common nowadays, occasionally epidemics may evolve explosively. The average age at presentation has risen and the likelihood of symptomatic infection is higher.