Diet-induced obesity reduces bone marrow T and B cells and promotes tumor progression in a transplantable Vk*MYC model of multiple myeloma.

Obesity is associated with an increased risk of developing multiple myeloma (MM). The molecular mechanisms causing this association is complex and incompletely understood. Whether obesity affects bone marrow immune cell composition in multiple myeloma is not characterized. Here, we examined the effect of diet-induced obesity on bone marrow immune cell composition and tumor growth in a Vk*MYC (Vk12653) transplant model of multiple myeloma. We find that diet-induced obesity promoted tumor growth in the bone marrow and spleen and reduced the relative number of T and B cells in the bone marrow. Our results suggest that obesity may reduce MM immune surveillance and thus may contribute to increased risk of developing MM.

Treadmill running intensity and post-exercise increase in plasma cardiac troponin I and T-A pilot study in healthy volunteers.

BACKGROUND: Plasma concentrations of cardiac troponins increase in healthy individuals after strenuous training, but the response to lower exercise intensities has not been characterized. AIM: To determine whether exercise at moderate intensity significantly increases plasma cardiac troponins measured with different assays in healthy recreational athletes. METHODS: Twenty-four self-reported healthy volunteers were instructed to complete three 60-min bouts of treadmill running at variable intensities: High-intensity training (HIT) including a maximal exercise test and an anaerobic threshold test followed by training at 80%-95% of maximum heart rate (HRmax ), Moderate-intensity training (MIT) at 60%-75% of HRmax , and Low-intensity training (LIT) at 45%-55% of HRmax . Blood samples were collected before and at 2, 4, and 6 h after HIT and 4 h after MIT and LIT. Troponin I and T were measured in plasma samples with assays from Abbot, Siemens, and Roche. RESULTS: Plasma troponins measured with all assays were significantly increased compared to baseline after HIT but not after LIT. After HIT, the fraction of all participants with one or more values above the assay-specific 99th percentiles ranged from 13% to 61%. The biomarker criteria for acute myocardial injury were met after HIT for troponin T in 75% of female participants having no clinical evidence of coronary artery disease. CONCLUSION: High-intensity, but not moderate- or low-intensity, training for 60 min induced a potentially clinically significant increase in plasma cardiac troponins in healthy volunteers. Results exceeding the population 99th percentiles were most frequent with the troponin T assay.

Reference change values of FIB-4.

When comparing two analytical results for the same analyte, the clinicians may benefit from knowing the reference change values (RCVs) of the analyte. For Fibrosis-4 Index (FIB-4), a noninvasive test used for assessing the risk of liver fibrosis, no RCVs have been published for non-cirrhotic individuals. Therefore, we estimated RCVs for adults, using retrospectively collected data from outpatients with AST, ALT, and thrombocytes within the respective reference intervals. FIB-4 was calculated as (age × AST)/(thrombocytes × ALT0.5). From two FIB-4 values in each patient we calculated the RCVs parametrically and non-parametrically. For both methods, we estimated the limits of the central 90% of the distribution of the ratio between the second and the first measurement. We obtained data on 599 outpatients with two blood tests taken 3 - 972 (median 258) days apart. The RCVs were 0.72 - 1.40 and 0.72 - 1.43, respectively, using the parametric and non-parametric methods. The 5 and 95 percentiles were not statistically significantly associated with sex, age, level of analyte, or the time between the measurements. The within-subject biological variation of FIB-4 was estimated to be 13.9%. Conclusion: In 90% of the patients the ratio between the second and the first FIB-4 result was approximately 0.7 - 1.4.

Calprotectin and CRP as biomarkers of cardiovascular disease risk in patients with chronic kidney disease: a follow-up study at 5 and 10 years.

Chronic kidney disease (CKD) and low-grade inflammation are associated with increased risk of cardiovascular disease (CVD). Calprotectin, a protein mainly secreted by activated neutrophils during inflammatory conditions, has been linked to CVD risk in general populations. The aim of this study was to evaluate the association of calprotectin with CVD risk in CKD patients, relative to C-reactive protein (CRP). One hundred and fifty-three patients with moderate CKD were prospectively followed up at 5 and 10 years. We used Cox regression modelling with stepwise adjustments for other relevant covariates (age, sex, cystatin C, previous CVD, systolic blood pressure, HDL cholesterol and HbA1c) to assess the association of baseline calprotectin and CRP with the risk of fatal or non-fatal CVD events. Twenty-nine and 44 patients experienced a CVD event during median follow-up of 4.8 and 10.9 years, respectively. Higher calprotectin was associated with increased CVD risk at both time points, which remained statistically significant after multivariable adjustments, including adjustment for CRP. For CRP, the associations did not remain statistically significant after final multivariable adjustments. In conclusion, we have shown that in patients with CKD, calprotectin was independently associated with the risk of future CVD events, suggesting that calprotectin may provide prognostic information of CVD risk.

Inflammatory markers and risk of cardiovascular mortality in relation to diabetes status in the HUNT study.

Inflammatory markers have been associated with increased risk of cardiovascular mortality in general populations. We assessed whether these associations differ by diabetes status. From a population-based cohort study (n = 62,237) we included all participants with diabetes (n = 1753) and a control group without diabetes (n = 1818). Cox regression models were used to estimate hazard ratios (HRs) with 95% confidence intervals (CI) for possible associations with cardiovascular mortality of 4 different inflammatory markers; C-reactive protein (CRP), calprotectin, neopterin and lactoferrin. During a median follow-up of 13.9 years, 728 (20.4%) died from cardiovascular disease (CVD). After adjustment for age, sex and diabetes, the associations of all inflammatory markers with risk of cardiovascular mortality were log-linear (all P ≤ 0.017 for trend) and did not differ according to diabetes status (all P ≥ 0.53 for interaction). After further adjustments for established risk factors, only CRP remained independently associated with cardiovascular mortality. HRs were 1.22 (1.12-1.32) per standard deviation higher loge CRP concentration and 1.91 (1.50-2.43) when comparing individuals in the top versus bottom quartile. The associations of CRP, calprotectin, lactoferrin and neopterin with cardiovascular mortality did not differ by diabetes, suggesting that any potential prognostic value of these markers is independent of diabetes status.

Using blood calprotectin as a measure of blood neutrophils.

Presently, bed-side or at home quantification of neutrophils in blood (b-neutrophils) is not practical, because cytometric methods are too expensive and technically demanding. We have explored whether calprotectin concentration in whole blood (b-calprotectin) might be a valid measure of b-neutrophils because this principle might be used in a simple and robust immunoassay device. We obtained heparin blood samples from 77 patients with possible neutropenia, most of them cancer patients treated with cytostatic drugs, and compared b-calprotectin with their b-neutrophils in a simultaneously taken EDTA-blood sample. The Spearman rank correlation coefficient between b-calprotectin and b-neutrophils was 0.986 (p < .0001). In a regression model of b-neutrophils as a function of age, gender, type of hematology instrument, total leukocyte count minus neutrophils, b-calprotectin, and plasma calprotectin (p-calprotectin), only b-calprotectin was a statistically significant predictor. B-neutrophils below 1 × 109/L was unlikely if b-calprotectin was above 50 mg/L. In conclusion, b-calprotectin, without adjusting for p-calprotectin, correlates closely with b-neutrophils and could be used to detect b-neutrophils below 1 × 109/L.

Diagnosing empty iron stores in women: unbound iron binding capacity (UIBC) versus soluble transferrin receptor (sTFR).

Unbound iron binding capacity (UIBC) is more accurate than total iron binding capacity (TIBC) and percent transferrin saturation in diagnosing empty iron stores. It is unknown whether UIBC is more or less accurate than soluble transferrin receptor (sTFR). We obtained public-use data from the U.S. National Health and Nutrition Examination Survey (NHANES) 2005-2006 to compare the accuray of UIBC and sTFR in diagnosing empty iron stores in 2337 women aged 12-49 years. We grouped the women according to CRP less than 5 mg/L and pregnancy (four groups) and used three definitions of empty iron stores: Serum ferritin less than 10, 15, and 20 µg/L. Receiver operating characteristic (ROC) curve analysis was used to estimate the diagnostic accuracy. UIBC showed a better diagnostic accuracy than sTFR in all groups and definitions of empty iron stores, except in nonpregnant women with CRP at least 5 mg/L when empty iron stores were defined as ferritin less than 10 and 15 µg/L. Two differences reached statistical significance: In nonpregnant women without inflammation the area under the ROC curve for UIBC was 0.830 compared to 0.793 for sTFR (p = .007) when empty iron stores were defined as ferritin less than 20 µg/L. The corresponding figures for pregnant women without inflammation were 0.843 for UIBC and 0.739 for sTFR (p = .003). In conclusion, UIBC is a more accurate test than sTFR in diagnosing empty iron stores in women without inflammation.

Establishing the 99th percentile of a novel assay for high-sensitivity troponin I in a healthy blood donor population.

Background: The recommended cut-off of cardiac troponin (cTn) for the diagnosis of acute myocardial infarction (AMI) is the 99th percentile in a healthy reference population. We aimed to determine the 99th percentile of the novel ADVIA Centaur® High Sensitivity Troponin I assay (Siemens Healthcare Diagnostics) in fresh lithium heparin plasma samples from healthy blood donors. Methods: A total of 1000 apparently healthy blood donors were included. High-sensitivity (hs) cTnI, hs-cTnT, creatinine and N-terminal pro b-type natriuretic peptide (NT-proBNP) were measured in fresh lithium heparin plasma samples, and glycated hemoglobin (HbA1c) was measured in ethylenediaminetetraacetic acid (EDTA)-blood. The 99th percentile was estimated for the whole population, as well as for males and females separately. Results: For the total population the 99th percentile of ADVIA Centaur® High Sensitivity Troponin I was 96 (65-149) ng/L. The estimated value differed significantly from results published by others and was highly dependent on which values were considered statistical outliers. Conclusions: The estimated 99th percentile for hs-cTnI in the population studied differed significantly from previously published results. There is a need for further specifications regarding how subjects used for estimating the 99th percentile of cTns in healthy populations should be recruited and how outlier values should be identified, as this can highly influence the diagnostic cut-off applied for AMI.

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.

Using probit regression to disclose the analytical performance of qualitative and semi-quantitative tests.

BACKGROUND: The analytical performance of qualitative and semi-quantitative tests is usually studied by calculating the fraction of positive results after replicate testing of a few specimens with known concentrations of the analyte. We propose using probit regression to model the probability of positive results as a function of the analyte concentration, based on testing many specimens once with a qualitative and a quantitative test. METHODS: We collected laboratory data where urine specimens had been analyzed by both a urine albumin ('protein') dipstick test (Combur-Test strips) and a quantitative test (BN ProSpec System). For each dipstick cut-off level probit regression was used to estimate the probability of positive results as a function of urine albumin concentration. We also used probit regression to estimate the standard deviation of the continuous measurement signal that lies behind the binary test response. Finally, we used probit regression to estimate the probability of reading a specific semi-quantitative dipstick result as a function of urine albumin concentration. RESULTS: Based on analyses of 3259 specimens, the concentration of urine albumin with a 0.5 (50%) probability of positive result was 57 mg/L at the lowest possible cut-off limit, and 246 and 750 mg/L at the next (higher) levels. The corresponding standard deviations were 29, 83, and 217 mg/L, respectively. Semi-quantitatively, the maximum probability of these three readings occurred at a u-albumin of 117, 420, and 1200 mg/L, respectively. CONCLUSIONS: Probit regression is a useful tool to study the analytical performance of qualitative and semi-quantitative tests.

Using the hazard ratio to evaluate allowable total error in predictive measurands.

BACKGROUND: Allowable total error is usually derived from data on biological variation or from state-of-the-art of measuring technology. Here we present a new principle for evaluating allowable total error when the concentration of the analyte (the measurand) is used for prediction: What are the predictive consequences of allowable total errors in terms of errors in the estimate of the hazard ratio (HR)? METHODS: We explored the effect of analytical measurement errors on Cox regression estimates of HR. Published data on Cox regression coefficients were used to illustrate the effect of measurement errors on predicting cardiovascular events or death based on serum concentration of cholesterol and on progression of chronic kidney disease to kidney failure based on serum concentrations of albumin, bicarbonate, calcium and phosphate, and urine albumin/creatinine-ratio. RESULTS: If the acceptable error in the estimate of the HR is 10%, allowable total errors in serum cholesterol, bicarbonate and phosphate are approximately the same as allowable total error based on biological variation, while allowable total error in serum albumin and calcium are a little larger than estimates based on biological variation. CONCLUSIONS: Evaluating allowable total error from its effect on the estimate of HR is universally applicable to measurands used for prediction.

Arginine, dimethylated arginine and homoarginine in relation to cardiovascular risk in patients with moderate chronic kidney disease.

OBJECTIVES: Arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), arginine/ADMA ratio and homoarginine could potentially affect nitric oxide production and have been studied in relation to cardiovascular risk (CVR) in various clinical populations. Prospective studies on the CVR associated with arginine/ADMA ratio and homoarginine in patients with moderate chronic kidney disease (CKD) are still scarce. We have studied how arginine, homoarginine and dimethylated arginine can predict cardiovascular events in such a population. DESIGN AND METHODS: We measured plasma concentrations of arginine (P-arginine), ADMA (P-ADMA), SDMA (P-SDMA), homoarginine (P-homoarginine) and other covariates in 160 patients with predialytic CKD (mean age 57 years and mean eGFR 43 mL/min/1.73 m(2)) and followed them for 58 months in median. The risks of fatal and non-fatal cardiovascular events associated with the predictors were evaluated with multivariable Cox proportional hazard analysis. RESULTS: There were 31 cardiovascular events during the observation period. In a multivariable model adjusted for age, sex, previous cardiovascular disease, P-cystatin C and P-homoarginine, the hazard ratio (HR) associated with an increase in arginine/ADMA ratio by 10 was 0.83 (P=0.03). The HR of a 1 µmol/L increase in P-homoarginine in the same model was 1.78 (P=0.01). A statistically significant interaction between P-homoarginine and P-cystatin C was found in an extended multivariable model. P-SDMA was not associated with increased CVR after adjustment for basic covariates. CONCLUSION: This study demonstrates a negative association between arginine/ADMA ratio and CVR in CKD patients and a positive association between P-homoarginine and CVR. The latter is in contrast to what has been demonstrated by others.