Rosalia Aloe

Italian multicentre study for application of a diagnostic algorithm in autoantibody testing for autoimmune rheumatic disease: conclusive results.

AIM The presence of specific auto-antibodies in serum (i.e., antinuclear antibodies or ANA, anti-extractable nuclear antigens or anti-ENA, and anti-double stranded DNA or anti-dsDNA ) is one of the major criteria in the diagnostics of Autoimmune Rheumatic Disease. As such, the request for these tests has grown exponentially in laboratory practice. The aim of this study is to describe the implementation of a joint laboratory-clinics guideline for reducing clinically inappropriate requests for autoantibody testing in a broad geographic area (Parma, Modena, Piacenza, Reggio-Emilia) for the diagnosis of Autoimmune Rheumatic Disease. METHODS This study, supported by a Regional grant for innovative research projects started in January 2008, is an observational research aimed at comparing the number of ANA, anti-dsDNA and anti-ENA testing as well as the percentage of positive test results before and after implementation of the diagnostic algorithm in hospitalized patients. A multidisciplinary team consisting of clinical immunologist and laboratory scientists was established, with the aim of collecting and analysing diagnostic criteria, clinical needs, laboratory report formats, analytical procedures, as well as the number of tests performed. The laboratory results and the clinical protocol were both validated by data emerging from the clinical follow-up studies. RESULTS A joint guideline for auto-antibody testing, placing ANA test at the first level, has been developed and implemented since January 2009. The results for the period January-June 2009 (12,738 tests) were compared with those of the same period in 2008 (13,067 tests). A significant reduction in the number of anti-dsDNA (-26%) and anti-ENA (-15%) was observed. The percentage of second-level tests positivity after implementation of the diagnostic protocol had also consistently increased for both ENA (13% vs 17%) and dsDNA (9% vs 11%). DISCUSSION The development and implementation of algorithms for the diagnostics of Autoimmune Rheumatic Disease in hospitalized patients was associated with a reduction in the number of second-level tests, but also with an increased diagnostic specificity. This outcome attests that close collaboration and audit between clinicians, laboratory specialists and healthcare services is effective to develop efficient diagnostic algorithms for both hospitalized patients and outpatients.

Human chorionic gonadotropin in pregnancy diagnostics.

Human chorionic gonadotropin (hCG) is a 237 aminoacid glycoprotein hormone composed of two dissimilar α and β subunits noncovalently linked by charge interactions, which are both required for the biological activity of the hormone. Due to structural heterogeneity, hCG exists in biological fluids as a mixture of different isoforms, i.e., intact active hormone (hCG), nicked hCG (hCGn), free β subunits (hCGβ), free α subunit (hCGα), β-core fragment (hCGβcf, predominantly detected in urine and containing amino acids 6-40 and 55-92 linked by disulphide bridges) and nicked free β-subunit (hCGβn). Although the measurement of hCG might be useful in a kaleidoscope of clinical conditions, such as diagnosis, monitoring and follow-up of pregnancy-related disorders, prenatal screening and gynecological cancers, the leading application is still the diagnosis of pregnancy, where it can be measured quantitatively either in serum or urine, in the latter case also using qualitative and rapid immunoassays. Since there is still debate as to whether serum or urine tests are to be preferred for establishing a diagnosis of pregnancy, we discuss here the main analytical and clinical aspects of hCG measurement for the diagnosis of pregnancy, highlighting the advantages and limitations of assessing hCG in urine and serum.

Evaluation of NGAL Test ™ , a fully-automated neutrophil gelatinase-associated lipocalin (NGAL) immunoassay on Beckman Coulter AU 5822

Abstract Background: The neutrophil gelatinase associated lipocalin (NGAL) has been identified as the most promising biomarker of acute kidney injury (AKI). This study was aimed to evaluate a NGAL immunoassay on Beckman Coulter AU 5822. Methods: NGAL Test™ (BioPorto Diagnostics A/S) is a particle-enhanced turbidimetric immunoassay. The within-and between-run imprecision were assessed on three urine samples. The linearity was assessed by serially diluting two urine samples with low and high NGAL concentration. The comparison study was performed with Abbott ARCHITECT NGAL, on 70 urine samples. Results: The within-run imprecision was comprised between 1.0% and 2.3%, whereas the between-run imprecision was between 1.2% and 2.0%. The linearity was excellent in the range between 18 ng/mL and 790 ng/mL (r=1.000; p<0.001). A highly significant agreement was observed between NGAL Test™ on Beckman Coulter AU5822 and Abbott ARCHITECT NGAL (r=0.925; p<0.001), although the method exhibited a bias of +65%. Excellent sensitivity and specificity against the ARCHITECT values were found at 200 ng/mL. Conclusions: This analytical evaluation attests that the NGAL Test™ has several technical and analytical advantages, including no manual pretreatment, low volume of sample (i.e., 3 μL), fast turnaround time (approx. 10 min), low imprecision, wide dynamic range, optimal linearity.

Serum levels of protein S100B predict intracranial lesions in mild head injury

OBJECTIVES This study was aimed to assess whether serum S100B levels at emergency department admission can be used to omit unnecessary computed tomography (CT) in patients with minor head injury (MHI). DESIGN AND METHODS Sixty consecutive patients with recent MHI were included in this study. Serum S100B measurement and CT scanning were performed in all patients within 3h from head injury. RESULTS A positive CT scan was present in 20 out of 60 subjects. Significantly higher values of protein S100B were found in CT positive than in CT negative patients (1.35 versus 0.48 μg/L; p<0.001). The area under the ROC curve for protein S100B was highly significant (AUC 0.80; p<0.001) and a S100B cut-off value of 0.38 μg/L displayed 100% sensitivity and 58% specificity. CONCLUSIONS Serum S100-B levels might allow to omit unnecessary CT in patients with pure MHI, thus reducing radiation exposure and saving healthcare resources.

Troponin I measured with a high sensitivity immunoassay is significantly increased after a half marathon run.

Abstract Only a few studies have assessed the kinetics of cardiac troponins after endurance exercise by using the novel high-sensitive (HS) immunoassays. These were based on the measurement of HS-TroponinT (TnT), but not HS-Troponin I (TnI), and exclusively involved marathon or ultra-marathon contests. TnI was measured in 17 healthy trained Caucasian males performing a 21 km, half-marathon, with the conventional AccuTnI and the HS-AccuTnI immunoassays. The concentration of HS-AccuTnI significantly increased from the mean baseline value of 2.9 ng/L (Interquartile range [IQR], 2.4– 4.3 ng/L) to 4.8 (IQR, 3.0–5.7 ng/L) after the run (p = 0.002), 9.0 ng/L (IQR, 5.8–15.3 ng/L) at 3h (p < 0.001), 12.3 ng/L (IQR, 7.1–21.5 ng/L) at 6h (p < 0.001), and 4.5 ng/L (IQR, 2.8–6.0 ng/L) at 24 h (p = 0.003) afterwards. The variation throughout the study period was statistically significant (p < 0.001). Age, training history, finishing time and exercise intensity were not associated with changes of HS-AccuTnI. The values of the TnI measured with the conventional AccuTnI immunoassay were always below the 99th percentile reference limit, except in one subject 3 h after the run, and in two subjects 6 h after the run. These results attest that TnI values measured with the novel HS-AccuTnI immunoassay were significantly increased in athletes participating in a half marathon.

Erythrocyte mechanical fragility is increased in patients with type 2 diabetes

BACKGROUND Anemia is common among patients with type 2 diabetes. We determined whether type 2 diabetic patients significantly differed in erythrocyte mechanical fragility as compared with nondiabetic subjects. METHODS We recruited 25 Caucasian patients with type 2 diabetes (14 men and 11 women; mean age 58±8 years) and 25 age-, race- and sex-matched nondiabetic individuals. The fragility of erythrocytes was tested by inducing mechanical hemolysis by double aspiration of K(2)EDTA blood through a 0.5 mL insulin syringe equipped with a very thin needle. The plasma was then separated from the blood cells by centrifugation at 2000 xg for 15 min at room temperature. A Beckman Coulter DxC 800 was used to measure the hemolysis index by direct spectrophotometry. RESULTS Compared with matched nondiabetic controls, type 2 diabetic patients had a significantly increased mechanical fragility of erythrocytes (hemolysis index ratio 21±13 vs. 14±10, p=0.02). Univariable linear regression analysis revealed that there was a strong positive association between percent hemolysis and fasting plasma glucose (r=0.669, p<0.001) and hemoglobin A1c (r=0.549, p<0.005) in type 2 diabetic subjects, but not in matched nondiabetic controls. CONCLUSIONS Our data suggest that patients with type 2 diabetes have a significantly higher erythrocyte mechanical fragility than matched nondiabetic subjects, and that fasting plasma glucose is the strongest correlate of increased mechanical fragility of erythrocytes in this patients group.

Variation of serum and urinary neutrophil gelatinase associated lipocalin (NGAL) after strenuous physical exercise

Abstract Background: Strenuous exercise may trigger acute complications, such as exertional rhabdomyolysis and gastrointestinal complaint. As less is known about the potential renal impairment after long distance running, we assessed creatinine and neutrophil gelatinase associated lipocalin (NGAL) in serum (sNGAL) and urine (uNGAL) before and after an ultramarathon. Methods: The study population consisted of 16 trained male athletes who ran a 60 km ultramarathon. Blood and spot urine samples were collected 20 min before and immediately after the run. Creatinine was assessed by Jaffe assay on Beckman Coulter AU5800 and renal function was expressed as estimated glomerular filtration rate (eGFR) by MDRD formula. NGAL was measured by fully-automated immunoassay NGAL Test™ on AU 5800. Results: Serum and urinary creatinine increased significantly by 38% and 78%, respectively. The eGFR contextually decreased by 31%. sNGAL, uNGAL and uNGAL/creatinine ratio increased by 1.6-fold, 7.7-fold and 2.9-fold. In six of 16 athletes (38%), the acute post-exercise increase of serum creatinine met the criteria of acute kidney injury. No significant relationship was found between pre-exercise, post-exercise values and post-exercise variation of sNGAL, uNGAL and uNGAL/creatinine ratio. A significant correlation was found between pre- and post-exercise changes of serum creatinine and sNGAL, but not with either uNGAL or uNGAL/creatinine ratio. Conclusions: The acute variations of serum creatinine and uNGAL attest that renal impairment is likely to develop after long distance running. The uNGAL seems more independent from creatinine variation and extra-renal sources, and thereby more reliable for monitoring the renal involvement in these types of kidney impairment.

Foot-strike haemolysis after a 60-km ultramarathon

BACKGROUND The various contributors to sport-related anaemia include increased plasma volume, exercise-induced oxidative stress, increased body temperature, acidosis, gastrointestinal bleeding, acute and chronic inflammation as well as compression and damage of red blood cells (RBC) in the capillaries within the contracting muscles. The effective contribution of foot-strike haemolysis is unclear. MATERIALS AND METHODS We studied 18 Caucasian male athletes (mean age, 42 years; range, 34-52 years) before and immediately after a 60-km ultramarathon. Laboratory investigations included the haematological profile along with haptoglobin, potassium, aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH) and albumin concentrations and a haemolysis index (HI). RESULTS No significant variations were found in post-exercise values of haemoglobin, RBC count and haematocrit. Mean corpuscular volume and haptoglobin were significantly decreased, whereas RBC distribution width was increased. The concentration of haptoglobin was reduced by approximately 50%, whereas enzyme concentrations were all remarkably increased. The HI remained below 0.5 g/L. After adjusting for plasma volume change, the increases were 1.7% for potassium (P=0.17), 30% for AST (P<0.01), 49% for LDH (P<0.01) and 2.39-fold for CK (P<0.01). A statistically significant association was found between haemoconcentration-adjusted variations of CK and those of AST (r=0.803; P<0.01) and LDH (r=0.551; P=0.02). DISCUSSION This is the first study demonstrating that long-distance running does not induce clinically significant changes in haemoglobin, haematocrit, RBC count or potassium concentration. The significant post-exercise decrease of haptoglobin reflects a certain degree of haemolysis, but the concentration of cell-free haemoglobin remaining below 0.5 g/L and the non-significant variation in RBC count both indicate that the foot-strike haemolysis is very modest or even clinically negligible.

Influence of in vitro hemolysis on hematological testing on Advia 2120

Introduction:  Although there is broad knowledge on the effect of several preanalytical errors on laboratory hematology, there is no information on the reliability of routine hematological testing on hemolyzed specimens.

Influence of hemolysis on troponin testing: studies on Beckman Coulter UniCel Dxl 800 Accu-TnI and overview of the literature

Abstract Background: Hemolyzed specimens are the leading pre-analytical problem in the laboratory practice, and exert a negative impact on test results. We assessed the reliability of Beckman Coulter UniCel Dxl 800 Accu-TnI testing on hemolyzed specimens. Methods: Twelve non-hemolyzed K2EDTA-anticoagulated samples displaying Accu-TnI values >0.20 μg/L and nine with values <0.04 μg/L were selected and three aliquots were obtained from each. The first (“#A”) was processed without further manipulation, whereas the second (“#B”) and third (“#C”) were hemolyzed by aspirating anticoagulated blood through a thin needle. Plasma was separated and tested for hemolysis index (HI) and Accu-TnI. Results: As compared with aliquot #A (HI: 0), a progressive increase of hemolysis occurred in aliquots #B (HI: 25) and #C (HI: 45). The concentration of Accu-TnI gradually decreased from aliquots #A (0.89 μg/L, 0.20–20.16 μg/L), to aliquots #B (0.81 μg/L, 95% CI 0.17–18.37 μg/L; p=0.041) and #C (0.78 μg/L, 95% CI 0.15–17.48 μg/L; p=0.026). In 0/12 (aliquots #B) and 3/12 cases (aliquots #C) the percent decrease exceeded 20% variation. The values remained unchanged in nine samples with Accu-TnI <0.04 μg/L. Conclusions: Accu-TnI values decrease in hemolyzed samples, but the bias might not be clinically significant in samples with hemoglobin <14.5 g/L.