Thrombopoietin/TGF-ß1 Loop Regulates Megakaryocyte Extracellular Matrix Component Synthesis.

Extracellular matrix (ECM) components initiate crucial biochemical and biomechanical cues that are required for bone marrow homeostasis. In our research, we prove that a peri-cellular matrix composed primarily of type III and type IV collagens, and fibronectin surrounds human megakaryocytes in the bone marrow. The data we collected support the hypothesis that bone marrow megakaryocytes possess a complete mechanism to synthesize the ECM components, and that thrombopoietin is a pivotal regulator of this new function inducing transforming growth factor-ß1 (TGF-ß1) release and consequent activation of the downstream pathways, both in vitro and in vivo. This activation results in a dose dependent increase of ECM component synthesis by megakaryocytes, which is reverted upon incubation with JAK and TGF-ß1 receptor specific inhibitors. These data are pivotal for understanding the central role of megakaryocytes in creating their own regulatory niche within the bone marrow environment.

A staging system for renal outcome and early markers of renal response to chemotherapy in AL amyloidosis.

The kidney is involved in 70% of patients with immunoglobulin light-chain (AL) amyloidosis, but little is known on progression or reversibility of renal involvement, and criteria for renal response have never been validated. Newly diagnosed patients from the Pavia (n = 461, testing cohort) and Heidelberg (n = 271, validation cohort) centers were included. Proteinuria >5 g/24 h and estimated glomerular filtration rate (eGFR) <50 mL/min predicted progression to dialysis best. Proteinuria below and eGFR above the thresholds indicated low risk (0 and 4% at 3 years in the testing and validation cohorts, respectively). High proteinuria and low eGFR indicated high risk (60% and 85% at 3 years). At 6 months, a ≥25% eGFR decrease predicted poor renal survival in both cohorts and was adopted as criterion for renal progression. A decrease in proteinuria by ≥30% or below 0.5 g/24 h without renal progression was the criterion for renal response, being associated with longer renal survival in the testing and validation populations. Hematologic very good partial or complete remission at 6 months improved renal outcome in both populations. We identified and validated a staging system for renal involvement and criteria for early assessment of renal response and progression in AL amyloidosis that should be used in clinical practice and trial design.

Discoidin domain receptor 1 protein is a novel modulator of megakaryocyte-collagen interactions.

Growing evidence demonstrates that extracellular matrices regulate many aspects of megakaryocyte (MK) development; however, among the different extracellular matrix receptors, integrin α2ß1 and glycoprotein VI are the only collagen receptors studied in platelets and MKs. In this study, we demonstrate the expression of the novel collagen receptor discoidin domain receptor 1 (DDR1) by human MKs at both mRNA and protein levels and provide evidence of DDR1 involvement in the regulation of MK motility on type I collagen through a mechanism based on the activity of SHP1 phosphatase and spleen tyrosine kinase (Syk). Specifically, we demonstrated that inhibition of DDR1 binding to type I collagen, preserving the engagement of the other collagen receptors, glycoprotein VI, α2ß1, and LAIR-1, determines a decrease in MK migration due to the reduction in SHP1 phosphatase activity and consequent increase in the phosphorylation level of its main substrate Syk. Consistently, inhibition of Syk activity restored MK migration on type I collagen. In conclusion, we report the expression and function of a novel collagen receptor on human MKs, and we point out that an increasing level of complexity is necessary to better understand MK-collagen interactions in the bone marrow environment.

The importance of calcium in the regulation of megakaryocyte function.

Platelet release by megakaryocytes is regulated by a concert of environmental and autocrine factors. We previously showed that constitutively released adenosine diphosphate by human megakaryocytes leads to platelet production. Here we show that adenosine diphosphate elicits, in human megakaryocytes, an increase in cytosolic calcium concentration, followed by a plateau, which is lowered in the absence of extracellular calcium, suggesting the involvement of Store-Operated Calcium Entry. Indeed, we demonstrate that megakaryocytes express the major candidates to mediate Store-Operated Calcium Entry, stromal interaction molecule 1, Orai1 and canonical transient receptor potential 1, which are activated upon either pharmacological or physiological depletion of the intracellular calcium pool. This mechanism is inhibited by phospholipase C or inositol-3-phosphate receptor inhibitors and by a specific calcium entry blocker. Studies on megakaryocyte behavior, on extracellular matrix proteins that support proplatelet extension, show that calcium mobilization from intracellular stores activates signaling cascades that trigger megakaryocyte adhesion and proplatelet formation, and promotes extracellular calcium entry which is primarily involved in the regulation of the contractile force responsible for megakaryocyte motility. These findings provide the first evidence that both calcium mobilization from intracellular stores and extracellular calcium entry specifically regulate human megakaryocyte functions.

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A.

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood.

The combination of high-sensitivity cardiac troponin T (hs-cTnT) at presentation and changes in N-terminal natriuretic peptide type B (NT-proBNP) after chemotherapy best predicts survival in AL amyloidosis.

In light-chain (AL) amyloidosis, prognosis is dictated by cardiac dysfunction. N-terminal natriuretic peptide type B (NT-proBNP) and cardiac troponins (cTn) are used to assess the severity of cardiac damage. We evaluated the prognostic relevance of a high-sensitivity (hs) cTnT assay, NT-proBNP, and cardiac troponin I in 171 consecutive patients with AL amyloidosis at presentation and 6 months after treatment. Response and progression of NT-proBNP were defined as more than 30% and more than 300 ng/L changes. All 3 markers predicted survival, but the best multivariable model included hs-cTnT. The hs-cTnT prognostic cutoff was 77 ng/L (median survival 10.6 months for patients with hs-cTnT above the cutoff). After treatment, response and progression of NT-proBNP and a more than 75% increase of hs-cTnT were independent prognostic determinant. In AL amyloidosis, hs-cTnT is the best baseline prognostic marker. Therapy should be aimed at preventing progression of cardiac biomarkers, whereas NT-proBNP response confers an additional survival benefit.

Best use of cardiac biomarkers in patients with AL amyloidosis and renal failure.

In AL amyloidosis prognosis depends on the severity of heart dysfunction which is best assessed by natriuretic peptides (BNP and NT-proBNP). However, their clearance relies on glomerular filtration rate (GFR) and their concentration increases with renal failure. We evaluated the diagnostic and prognostic performance of NT-proBNP and BNP in 248 patients with AL amyloidosis with different degrees of renal failure. Patients were grouped according to GFR. Group 1 comprised 109 patients with GFR ≥60 mL/min/1.73 m(2) , Group 2, 77 subjects with GFR <60 and ≥15 mL/min/1.73 m(2) , and Group 3, 62 patients with GFR <15 mL/min/1.73 m(2) . The ability of natriuretic peptides to detect heart involvement and to predict survival in the three groups was assessed. Decreasing eGFR required higher cutoffs of both NT-proBNP and BNP for detecting heart involvement and predicting survival. Both natriuretic peptides were independent prognostic markers in Groups 1 and 2, whereas in Group 3 only BNP independently predicted survival. Natriuretic peptides are powerful and useful markers of cardiac dysfunction and prognosis, provided that eGFR is considered in interpreting their clinical meaning. BNP should be preferred in patients with end-stage renal failure.

Component-resolved diagnosis for phleum allergy: the role of recombinants.

BACKGROUND: Conventional diagnostic tests (such as radioallergosorbent test [RAST] and skin prick test [SPT]) use native raw pollen allergen extracts to establish allergy. However, recombinant allergens may offer important advantages compared with their natural counterparts. OBJECTIVE: This study evaluated serum immunoglobulin E (IgE) in patients with grass-induced allergic rhinitis (AR) or AR with asthma (ARA), comparing assays with natural or recombinant grass allergens. METHODS: Sixty patients (33 AR, 27 ARA) positive with SPT and serum IgE for Phleum pratense were enrolled in the study. Serum IgE specific for conventional and recombinant Phleum pratense: rPhl p 1, rPhl p 2, nPhl p 4, rPhl 5b, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, were measured by the IFMA procedure (ImmunoCAP, Phadia, Uppsala, Sweden). Data were expressed as the median (md) and percentiles. Recombinant allergen results were expressed also as the percentage of positive concentrations. The Wilcoxon test was used to compare samples. Because diagnosis is a binary variable (AR/ARA), logistic regression analysis was performed to identify possible correlates. RESULTS: IgE concentrations assessed with recombinant allergens were significantly higher in ARA patients (p = .05) than in AR patients. A value >5.8 kU/L is the optimal cut-off to discriminate AR and ARA patients. Model specificity was 76%, sensitivity 78%, and efficiency 77%. CONCLUSION: This study shows that IgEs for natural and recombinant grass pollen allergens are significantly higher in patients with AR and asthma. Moreover, using recombinant allergens it is possible to define a prediction model for diagnosis with 77% efficiency. Therefore, this study may suggest that there are advantages of using recombinant or purified, native allergens over crude extracts.

Identification of amyloidogenic light chains requires the combination of serum-free light chain assay with immunofixation of serum and urine.

BACKGROUND: The diagnosis of systemic immunoglobulin light-chain (AL) amyloidosis requires demonstration of amyloid deposits in a tissue biopsy and amyloidogenic monoclonal light chains. The optimal strategy to identify the amyloidogenic clone has not been established. We prospectively assessed the diagnostic sensitivity of the serum free light chain (FLC) kappa/lambda ratio, a commercial serum and urine agarose gel electrophoresis immunofixation (IFE), and the high-resolution agarose gel electrophoresis immunofixation (HR-IFE) developed at our referral center in patients with AL amyloidosis, in whom the amyloidogenic light chain was unequivocally identified in the amyloid deposits. METHODS: The amyloidogenic light chain was identified in 121 consecutive patients with AL amyloidosis by immunoelectron microscopy analysis of abdominal fat aspirates and/or organ biopsies. We characterized the monoclonal light chain by using IFE and HR-IFE in serum and urine and the FLC kappa/lambda ratio in serum. We then compared the diagnostic sensitivities of the 3 assays. RESULTS: The HR-IFE of serum and urine identified the amyloidogenic light chain in all 115 patients with a monoclonal gammopathy. Six patients with a biclonal gammopathy were omitted from the statistical analysis. The diagnostic sensitivity of commercial serum and urine IFE was greater than that of the FLC kappa/lambda ratio (96% vs 76%). The combination of serum IFE and the FLC assay detected the amyloidogenic light chain in 96% of patients. The combination of IFE of both serum and urine with the FLC kappa/lambda ratio had a 100% sensitivity. CONCLUSIONS: The identification of amyloidogenic light chains cannot rely on a single test and requires the combination of a commercially available FLC assay with immunofixation of both serum and urine.

Treatment of patients with advanced cardiac AL amyloidosis with oral melphalan, dexamethasone, and thalidomide.

Patients with primary (AL) amyloidosis and heart failure have a very poor prognosis and cannot tolerate aggressive therapy, such as autologous stem cell transplantation and high-dose dexamethasone-based regimens. We prospectively treated 22 patients with advanced cardiac amyloidosis combining oral melphalan, thalidomide, and reduced intensity dexamethasone (MTD). Six patients died due to cardiac amyloidosis before completing cycle 3. Early death was associated with reduced ejection fraction. Eight patients achieved a hematological response and four achieved a durable improvement of cardiac dysfunction. Treatment with MTD is feasible in patients with advanced cardiac AL amyloidosis and effective in subjects with preserved systolic function.

beta2-Microglobulin is potentially neurotoxic, but the blood brain barrier is likely to protect the brain from its toxicity.

BACKGROUND: In dialysis-related amyloidosis, beta2-microglobulin accumulates as amyloid fibrils preferentially around bones and tendons provoking osteoarthritis. In addition to the pathologic role played by the amyloid fibrils, it can be speculated that a pathogenic role is also played by the high concentrations of soluble beta2-microglobulin because it is toxic for certain cell lines like HL60 and mitogen for other cells such as the osteoclasts. The discovery that beta2-microglobulin can influence the biology of certain cells may lead to the assumption that it might affect neuronal cells that are quite sensitive to amyloidogenic proteins in the oligomeric state. Such a concern might be supported by clinical evidence that haemodialysis is associated with the risk of a cognitive impairment. METHODS: The cytotoxicity of beta2-microglobulin on the SH-SY5Y neuroblastoma cells was assayed by the MTT test. The beta2-microglobulin concentration was determined in the cerebrospinal fluid of four different patients by means of immunonephelometry and western blot. RESULTS: Oligomeric beta2-microglobulin is cytotoxic for the SH-SY5Y cells at a concentration that can be easily reached in the plasma of patients on haemodialysis. However, the beta2-microglobulin concentration, measured in the cerebrospinal fluid of a haemodialysis patient, appears to be independent of its plasma concentration and is maintained under the lower limit of cytotoxicity we have determined in the cell culture. CONCLUSIONS: Although beta2-microglobulin is potentially neurotoxic, it is unlikely that this protein plays a role in the pathophysiology of cognitive impairment observed in haemodialysis patients due to the protective effect of the blood brain barrier that maintains a low concentration of beta2-microglobulin in the cerebrospinal fluid.

AL amyloidosis associated with IgM monoclonal protein: a distinct clinical entity.

IgM-associated AL amyloidosis is rare and may represent a distinct entity. Sixty (7%) of 868 consecutive AL patients referred to our center had an IgM monoclonal protein. They were significantly older than non-IgM patients (median, 67 years vs. 62 years), had a higher frequency of lymph-node involvement (25% vs. 2%) and significantly lower median proteinuria (1.2 g/24h vs. 3.4 g/24h), N-terminal pro-natriuretic peptide type-B (1177 ng/L vs. 2135 ng/L) and troponin I (0.02 ng/mL vs. 0.05 ng/mL). In IgM patients, kappa light-chains were more frequent (42% vs. 23%) and the involved free light-chain concentration was lower (median 63 mg/L vs. 182 mg/L). Serum albumin and NT-proBNP were independent prognostic determinants. Response to treatment improved survival. The 14 patients who received melphalan/dexamethasone showed a 64% hematologic (complete remissions, 29%) and a 43% organ response rate. IgM-associated AL amyloidosis is a distinct entity, with less advanced organ dysfunction. Treatment with melphalan/ dexamethasone might be effective in these patients.

The impact of first-trimester serum free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A on the diagnosis of fetal growth restriction and small for gestational age infant.

OBJECTIVE: To evaluate the risk of fetal growth restriction (FGR) associated with first-trimester maternal serum concentrations of pregnancy-associated plasma protein A (PAPP-A) and free beta-human chorionic gonadotropin (beta-hCG). METHODS: A longitudinal study of 2,178 women who underwent first-trimester evaluation of serum PAPP-A and free beta-hCG. FGR was defined as a decrement of the fetal abdominal circumference to below the 10th percentile of our standard growth curve in the presence of Doppler signs of impaired placental perfusion. Logistic regression was used to compute multivariable odds ratios and the estimated prevalences of outcomes associated with first-trimester serum marker concentrations. RESULTS: The prevalences of small for gestational age (SGA, <10th percentile birth-weight) neonates and FGR were significantly higher among women with serum PAPP-A concentrations below the 10th percentile than in controls: 40/206 compared to 183/1,928, for SGA, adjusted odds ratio = 2.1, 95% confidence intervals (CI) 1.4-3.03; 24/75 compared to 182/1,900, for FGR, adjusted odds ratio = 3.9, 95% CI 2.3-6.5. The adjusted prevalences of FGR and SGA among women with simultaneous low first-trimester values of PAPP-A and free beta-hCG were 0.21 (95% CI 0.13-0.33) and 0.26 (95% CI 0.17-0.36), respectively. CONCLUSION: Low first-trimester maternal serum PAPP-A concentrations are significantly associated with reduced fetal size and increased risk of FGR with Doppler signs of impaired placental perfusion.

Predictive value of antibodies to citrullinated peptides and rheumatoid factors in anti-TNF-alpha treated patients.

This article will focus on the relationship between serum levels of anti-citrullinated peptide antibodies (anti-CCP) or rheumatoid factor (RF) and clinical response to TNF-alpha blockers in order to evaluate whether these antibodies may have a role as serological markers of response to therapy in rheumatoid arthritis (RA). The changes induced in anti-CCP levels after TNF blocking therapy still remain a controversial issue even though a marked reduction following conventional DMARDs has been reported in early disease. On the other hand, a drop in RF levels during treatment has been reported by many authors. Decreased IgM RF levels seem to parallel clinical response suggesting that this antibody can also be regarded as a marker of response to treatment. Pre-treatment RF positivity or negativity does not influence response to TNF-alpha blocking therapy while high pre-treatment levels of IgA RF seem to be associated with a poor response rate.

Clinical aspects of systemic amyloid diseases.

Amyloidosis is a protein misfolding disorder in which soluble proteins aggregate as insoluble amyloid fibrils. Protein aggregates and amyloid fibrils cause functional and structural organ damage respectively. To date, at least 24 different proteins have been recognized as causative agents of amyloid diseases, localized or systemic. The two most common forms of systemic amyloidosis are light-chain (AL) amyloidosis and reactive AA amyloidosis due to chronic inflammatory diseases. beta(2)-microglobulin amyloidosis is a common complication associated with long-term hemodialysis. Hereditary systemic amyloidoses are a group of autosomal dominant disorders caused by mutations in the genes of several plasma proteins. Heterogeneity in clinical presentation, pattern of amyloid-related organ toxicity and rate of disease progression is observed among systemic amyloidoses. In particular, beta(2)-microglobulin presents unique clinical features compared to the other systemic forms. The phenotypic features of hereditary systemic amyloidoses may instead overlap those of the two more common forms of acquired amyloidoses mentioned above and therefore a correct diagnosis can not rely only on clinical grounds. Unequivocal identification of the deposited protein is essential in order to avoid misdiagnosis and inappropriate treatment. Amyloid deposits can be reabsorbed and organ dysfunction reversed if the concentration of the amyloidogenic protein is reduced or zeroed. At present, the most effective approach to treatment of the systemic amyloidoses involves shutting down, or substantially reducing the synthesis of the amyloid precursor, or, as in the case of beta(2)-microglobulin, promoting its clearance.

Interferon-gamma- and interleukin-4-producing T cells in Down's syndrome.

Down's syndrome (DS) associates with genetic-dependent dysregulation of the interferon (IFN) system. We used intracellular cytokine staining to analyse the percentages of IFN-gamma- and interleukin (IL)-4-producing T cells in the peripheral blood of patients with DS, individuals with mental retardation (MR), and healthy controls (HCs). The percentages of IFN-gamma-producing CD4(+) and CD8(+) T cells (IFGCs), namely Th1 (mean, 21.4+/-S.D. 1.3) and Tc1 (12.6+/-1.1), and the Th1/Th2 ratio (6.1+/-0.2) in DS were significantly higher than in MR (15.9+/-1.3, 7.9+/-0.6, 4.8+/-0.3) and in HCs (15.6+/-1.9, 7.2+/-1.1, 4.6+/-0.6). Most of the DS patients with high IFGC percentages were seropositive for anti-transglutaminase IgA. We found no correlation between sex, age, APOE genotypes, coexisting autoimmune diseases, susceptibility to infections, or degree of cognitive impairment and high IFGC percentages. This abnormality might thus contribute to immune dysfunction in DS without manifest clinical correlates.

Serum N-terminal pro-brain natriuretic peptide is a sensitive marker of myocardial dysfunction in AL amyloidosis.

BACKGROUND: Brain natriuretic peptide (BNP) is a marker of ventricular dysfunction and can be used to assess prognosis in heart failure and after myocardial infarction. Heart involvement is the most important prognostic factor and causes death in almost all patients with light-chain amyloidosis (AL). We investigated the prognostic value of NT-proBNP and its utility in monitoring amyloid heart dysfunction. METHODS AND RESULTS: NT-proBNP was quantified at diagnosis in 152 consecutive patients seen at the coordinating center of the Italian Amyloidosis Study Group (Pavia) from 1999 throughout 2001. Heart involvement was estimated on the basis of clinical signs, electrocardiography, and echocardiography. NT-proBNP concentrations differed in patients with (n=90, 59%) and without (n=62, 41%) heart involvement (median: 507.8 pmol/L versus 22.1 pmol/L, P=10(-7)). The best cutoff for heart involvement was at 152 pmol/L (sensitivity: 93.33%, specificity: 90.16%, accuracy: 92.05%) and distinguished two groups with different survival (P<0.001). The Cox multivariate model including NT-proBNP was better than models including echocardiographic and clinical signs of heart involvement. NT-proBNP appeared to be more sensitive than conventional echocardiographic parameters in detecting clinical improvement or worsening of amyloid cardiomyopathy during follow-up. CONCLUSIONS: NT-proBNP appeared to be the most sensitive index of myocardial dysfunction and the most powerful prognostic determinant in AL amyloidosis. It adds prognostic information for newly diagnosed patients and can be useful in designing therapeutic strategies and monitoring response. NT-proBNP is a sensitive marker of heart toxicity caused by amyloidogenic light chains.

Pro-neoangiogenic cytokines (VEGF and bFGF) and anemia in solid tumor patients.

Using a commercial ELISA assay, we evaluated circulating VEGF and bFGF levels in 203 consecutive patients with solid tumors, and sought a correlation between them and with the grade of anemia. Serum VEGF values were within the normal range in 128 patients (63.05%), with a mean value of 675.04 pg/ml (median, 571.00; range, 0-2796.54). The analysis of VEGF values per tumor group did not provide any statistically significant difference. Regarding bFGF, 143 patients (70.44%) had measurable, and thus abnormal, bFGF values. Overall, mean bFGF serum value was 57.14 pg/ml (median, 8.30; range, 0-4334.71), with the highest bFGF levels found in breast carcinoma patients. As expected, a large number of our patients was fairly anemic, mean hemoglobin level being 11.47 g/dl (median, 11.30; range, 7.1-19.20), the lowest titers being observed in prostate carcinoma patients. No statistically significant correlation was found between serum VEGF and hemoglobin values (r=0.004) but a significant negative correlation was seen between serum bFGF and hemoglobin (r=-0.22, p<0.05). Considering the different tumor groups, a statistically significant negative correlation between bFGF and hemoglobin becomes even more apparent in the subgroup of renal carcinoma patients (r=-0.55, p<0.05). In conclusion, our results demonstrate that there is a statistically significant correlation between systemic hypoxia (evaluated in terms of hemoglobin levels) and circulating bFGF values, but not VEGF; this correlation may lead to therapeutic interventions.

Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection.

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 microm, 150 x 4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate-acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9+/-4.0% for non-deproteinized samples and 93.2+/-4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean+/-SD concentrations in the darkness period were 23.18+/-7.44 pg/ml for free melatonin and 82.5+/-36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23+/-2.22 and 7.40+/-5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01+/-2.31 and 28.55+/-6.95 pg/ml for the free and total fraction, respectively.

Advantages of the lognormal approach to determining reference change values for N-terminal propeptide B-type natriuretic peptide.

BACKGROUND: Serial measurement of NT-proBNP is performed routinely in the monitoring and assessment of the effectiveness of therapy in patients being treated for chronic heart failure (CHF). Intra-individual changes in NT-proBNP levels over time are compared typically to a reference change value (RCV) determined using either a standard [i.e., nested analysis of variance (nANOVA)] or a lognormal approach. The RCV defines the minimum percent change in serial analyte values that exceeds the percent change expected due to biological variation alone. Currently, there is no consensus on which approach (nANOVA or lognormal) to determining RCV is better. AIMS: Based on these considerations, we aimed to illustrate the impact of data transformation on the calculation of the biological variation of NT-proBNP and discuss the utility of logarithmic transformation in monitoring patients with heart failure. METHODS: 15 healthy subjects were enrolled after informed consent; 5 blood specimens were collected twice a week. Nested ANOVA from replicate analyses was applied to obtain components of biological variation, on the raw data and after data transformation. RESULTS: NT-proBNP distribution being highly skewed required data transformation. Natural log transformation yielded normalization. An example demonstrates that for untransformed values the RCV was overestimated for low concentrations of NT-proBNP and underestimated for higher concentrations. CONCLUSIONS: Log-transformed data are often used in the establishment of reference intervals for evaluating laboratory tests results in clinical practice, especially when the reference interval data are not Gaussian distributed. As log-normal approach is the best approach to determining RCV values we encourage its use assessing the clinical utility of NT-proBNP serial testing. We propose that the log-normal approach becomes the standard approach to determining RCV and replaces the use of nANOVA.