Recentrifugation of Lithium Heparin Gel Separator Tubes up to 8 h after Blood Collection Has No Relevant Influence on the Stability of 30 Routine Biochemical Analytes.

BACKGROUND: Venipuncture for the purpose of blood analysis is often performed at remote locations, and samples may be centrifuged locally to preserve the integrity of analytes. At the central laboratory, these tubes may be centrifuged again in the routine process. However, limited research shows that >1 centrifugation cycle of gel separator tubes causes significant changes in analytes, in particular troponin I and potassium. These preanalytical test changes are undesirable and may lead to errors in diagnosis and treatment of patients. METHODS: Ten volunteers donated blood in 10 lithium heparin gel tubes. Per volunteer, 5 tubes were centrifuged with Becton Dickinson centrifugation settings and 5 tubes with our local centrifugation settings. For each centrifugation setting, 1 tube was centrifuged directly after venipuncture; the second tube, directly after venipuncture and again after 4 h; the third tube, directly after venipuncture and again after 8 h; the fourth tube, 4 h after venipuncture; the last tube, 8 h after venipuncture. Thirty routine chemistry analyses were performed in plasma directly after the last centrifugation cycle. All tubes were kept at room temperature. Analytes were considered unstable when the mean percentage deviation exceeded the total allowable error. RESULTS: Except for calcium, which slightly exceeded the predefined total allowable error limit, all the investigated analytes remained stable up to 8 h after a second centrifugation cycle with both centrifugation settings. CONCLUSION: This study shows that recentrifugation up to 8 h after blood collection does not cause relevant deviations in test results and may be applied safely.

No effect of n-3 fatty acids supplementation on NT-proBNP after myocardial infarction: the Alpha Omega Trial.

BACKGROUND: heart failure is a major risk factor for cardiovascular mortality, for which n-3 fatty acids may have beneficial effects. We examined the effect of marine eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and plant-derived alpha-linolenic acid (ALA) on N-Terminal-pro Brain Natriuretic Peptide (NT-proBNP), a biomarker of heart failure. METHODS: we randomly assigned 4837 post-myocardial infarction patients, aged 60-80 years (82% men), to margarines supplemented with a targeted additional intake of 400 mg/day EPA and DHA, 2 g/day ALA, EPA-DHA plus ALA, or placebo for 40 months. In a random selection of 639 patients, NT-proBNP was determined both at baseline and at the end of follow-up. NT-proBNP was loge-transformed and analysed by type of treatment using analysis of covariance adjusting for baseline NT-proNBP. RESULTS: patients consumed on average 19.8 g margarine/day, providing an additional amount of 238 mg/day EPA with 158 mg/day DHA, 1.98 g/day ALA, or both, in the active-treatment groups. In the placebo group, the geometric mean level NT-proBNP increased from 245 ng/l (95%-confidence interval [CI]: 207-290) to 294 ng/l (95%-CI: 244-352) after 40 months (p = 0.001). NT-proBNP levels were not affected by ALA (+8% versus placebo; 95%-CI: -8% to +25%; p = 0.34), EPA-DHA (+2% versus placebo; 95%-CI: -14% to +18%; p = 0.78), nor EPA-DHA plus ALA (+9% versus placebo; 95%-CI: -8% to +25%; p = 0.31) treatment. CONCLUSIONS: supplementation with modest amounts of EPA-DHA, with or without ALA, did not have a significant effect on NT-proBNP levels in patients with a history of myocardial infarction.

Effect of omega-3 fatty acids on kidney function after myocardial infarction: the Alpha Omega Trial.

BACKGROUND AND OBJECTIVES: Kidney function gradually decreases with age, and myocardial infarction accelerates this deterioration. Omega-3 (n-3) fatty acids may slow down the decline of kidney function. The effect of marine and plant-derived n-3 fatty acids on kidney function in patients after myocardial infarction was examined. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In the Alpha Omega Trial, 2344 patients with history of myocardial infarction ages 60-80 years old (81% men) were randomized to one of four trial margarines. The patients received an additional targeted amount of 400 mg/d eicosapentaenoic acid and docosahexaenoic acid, 2 g/d α-linolenic acid, eicosapentaenoic acid-docosahexaenoic acid plus α-linolenic acid, or placebo for 40 months. Serum cystatin C and serum creatinine were assessed at baseline and after 40 months. Creatinine-cystatin C-based GFR was estimated with the Chronic Kidney Disease Epidemiology Collaboration equation. RESULTS: Patients consumed 19.9 g margarine/d, providing an additional 239 mg/d eicosapentaenoic acid with 159 mg/d docosahexaenoic acid, 1.99 g/d α-linolenic acid, or both in the active treatment groups. After 40 months, compared with baseline, mean (±SD) creatinine-cystatin C-based GFR was -6.9 (±12.6), -4.8 (±13.4), -6.2 (±12.8), and -6.0 (±13.0) ml/min per 1.73 m(2) in the placebo, eicosapentaenoic acid-docosahexaenoic acid, α-linolenic acid, and eicosapentaenoic acid-docosahexaenoic acid plus α-linolenic acid groups, respectively. After 40 months, in patients receiving eicosapentaenoic acid-docosahexaenoic acid compared with placebo, the decline in creatinine-cystatin C-based GFR was 2.1 less (95% confidence interval, 0.6 to 3.6; P<0.01) ml/min per 1.73 m(2); other comparisons were not statistical significant. Odds ratios (95% confidence intervals) of incident CKD (<60 ml/min per 1.73 m(2)) and rapid decline of kidney function (≥3 ml/min per year) for eicosapentaenoic acid-docosahexaenoic acid compared with placebo were 0.83 (0.58 to 1.18) and 0.85 (0.67 to 1.08), respectively. CONCLUSIONS: Long-term supplementation with 400 mg/d eicosapentaenoic acid-docosahexaenoic acid provides a small beneficial effect on kidney function in patients with a history of myocardial infarction.

Dynamics of peripheral blood lymphocyte subpopulations in the acute and subacute phase of Legionnaires' disease.

STUDY OBJECTIVE: Absolute lymphocytopenia is recognised as an important hallmark of the immune response to severe infection and observed in patients with Legionnaires' disease. To explore the immune response, we studied the dynamics of peripheral blood lymphocyte subpopulations in the acute and subacute phase of LD. METHODS AND RESULTS: EDTA-anticoagulated blood was obtained from eight patients on the day the diagnosis was made through detection of L. pneumophila serogroup 1 antigen in urine. A second blood sample was obtained in the subacute phase. Multiparametric flow cytometry was used to calculate lymphocyte counts and values for B-cells, T-cells, NK cells, CD4+ and CD8+ T-cells. Expression of activation markers was analysed. The values obtained in the subacute phase were compared with an age and gender matched control group. Absolute lymphocyte count (×109/l, median and range) significantly increased from 0.8 (0.4-1.6) in the acute phase to 1.4 (0.8-3.4) in the subacute phase. B-cell count showed no significant change, while T-cell count (×106/l, median and range) significantly increased in the subacute phase (495 (182-1024) versus 979 (507-2708), p = 0.012) as a result of significant increases in both CD4+ and CD8+ T-cell counts (374 (146-629) versus 763 (400-1507), p = 0.012 and 119 (29-328) versus 224 (107-862), p = 0.012). In the subacute phase of LD, significant increases were observed in absolute counts of activated CD4+ T-cells, naïve CD4+ T-cells and memory CD4+ T-cells. In the CD8+ T-cell compartment, activated CD8+ T-cells, naïve CD8+ T-cell and memory CD8+ T-cells were significantly increased (p<0.05). CONCLUSION: The acute phase of LD is characterized by absolute lymphocytopenia, which recovers in the subacute phase with an increase in absolute T-cells and re-emergence of activated CD4+ and CD8+ T cells. These observations are in line with the suggested role for T-cell activation in the immune response to LD.

The neutrophil-lymphocyte count ratio in patients with community-acquired pneumonia.

STUDY OBJECTIVE: The neutrophil-lymphocyte count ratio (NLCR) has been identified as a predictor of bacteremia in medical emergencies. The aim of this study was to investigate the value of the NLCR in patients with community-acquired pneumonia (CAP). METHODS AND RESULTS: Consecutive adult patients were prospectively studied. Pneumonia severity (CURB-65 score), clinical characteristics, complications and outcomes were related to the NLCR and compared with C-reactive protein (CRP), neutrophil count, white blood cell (WBC) count. The study cohort consisted of 395 patients diagnosed with CAP. The mean age of the patients was 63.4 ± 16.0 years. 87.6% (346/395) of the patients required hospital admission, 7.8% (31/395) patients were admitted to the Intensive Care Unit (ICU) and 5.8% (23/395) patients of the study cohort died. The NLCR was increased in all patients, predicted adverse medical outcome and consistently increased as the CURB-65 score advanced. NLCR levels (mean ± SD) were significantly higher in non-survivors (23.3 ± 16.8) than in survivors (13.0 ± 11.4). The receiver-operating characteristic (ROC) curve for NLCR predicting mortality showed an area under the curve (AUC) of 0.701. This was better than the AUC for the neutrophil count, WBC count, lymphocyte count and CRP level (0.681, 0.672, 0.630 and 0.565, respectively). CONCLUSION: Admission NLCR at the emergency department predicts severity and outcome of CAP with a higher prognostic accuracy as compared with traditional infection markers.

Increased circulating apoptotic lymphocytes in children with Down syndrome.

Down syndrome (DS) resembles immunodeficiency with increased infections, auto-immune diseases, and hematological malignancies. Until now, immunological studies in DS mainly focused on T-lymphocytes. We recently described a profound B-lymphocytopenia in children with DS. This could be caused by increased apoptosis. Therefore, we determined expression of flowcytometric markers for apoptosis [Annexin-V (AV) and propidium iodide (PI)] on peripheral lymphocytes in 72 children with DS and 32 age-matched controls (AMC). Within the total lymphocyte compartment, apoptosis was more pronounced in DS; it increased with age. Moreover, apoptosis was highest within the B-lymphocyte compartment which may be a contributing factor to the B-lymphocytopenia found in DS. Pediatr Blood Cancer 2012; 59: 1310-1312. © 2012 Wiley Periodicals, Inc.

Plasma concentration of von Willebrand factor predicts mortality in patients on chronic renal replacement therapy.

BACKGROUND: Traditional cardiovascular risk factors do not explain the high incidence of cardiovascular mortality and morbidity in patients with end-stage renal disease. A prothrombotic state could accelerate the process of vascular disease in these patients. METHODS: In this study, four platelet activation markers (NAP-2, P-selectin, GP1b and RANTES) and two endothelial cell activation markers (von Willebrand factor and its propeptide) were measured in 671 haemodialysis patients and 275 patients on continuous ambulatory peritoneal dialysis (PD). All were long-term dialysis patients. The risk of all-cause and cardiovascular mortality was assessed in relation to these markers after a mean follow-up time of 2.5 years. RESULTS: The von Willebrand factor showed a positive correlation with total mortality in the haemodialysis patients. In an unadjusted model, the hazard rate (HR) of total mortality was 2.4 [95% confidence interval (95% CI) 1.7-3.4] in the upper quartile of von Willebrand factor compared with the lowest quartile. It remained statistically significant (HR 1.8; 95% CI 1.2-2.6) after adjustment for traditional risk factors. In contrast, no significant correlation was found between von Willebrand factor levels and total mortality in PD patients. Finally, no relationship between platelet activation markers and total mortality was found in either the haemodialysis or the PD patients. CONCLUSION: It can be concluded that chronic endothelial cell activation, but not platelet activation, is related to all-cause mortality in end-stage renal disease patients on long-term dialysis.

A girl with autoimmune cytopenias, nonmalignant lymphadenopathy, and recurrent infections.

We describe a girl, now 9 years of age, with chronic idiopathic thrombocytopenic purpura, persistent nonmalignant lymphadenopathy, splenomegaly, recurrent infections, and autoimmune hemolytic anemia. Her symptoms partly fit the definitions of both autoimmune lymphoproliferative syndrome (ALPS) and common variable immunodeficiency disorders (CVIDs). Genetic analysis showed no abnormalities in the ALPS-genes FAS, FASLG, and CASP10. The CVID-associated TACI gene showed a homozygous polymorphism (Pro251Leu), which is found also in healthy controls.

B-lymphocyte reconstitution after repeated rituximab treatment in a child with steroid-dependent autoimmune hemolytic anemia.

We report the detailed long-term reconstitution of B-lymphocyte subpopulations, immunoglobulins, and specific antibody production after two courses of rituximab in a young, previously healthy girl with steroid-dependent autoimmune hemolytic anemia. B-lymphocyte subpopulations were surprisingly normal directly after reconstitution. However, there was a slower reconstitution after the second rituximab course, especially of non-switched and switched memory B-lymphocytes, and a temporary decline in IgM below age-matched reference values.

Down syndrome B-lymphocyte subpopulations, intrinsic defect or decreased T-lymphocyte help.

Down syndrome (DS) is known for increased incidence of respiratory infections and autoimmune diseases, indicating impaired immunity. Until now, attention has been mainly focused on T lymphocytes. Therefore, we determined B-lymphocyte subpopulations in 95 children with DS compared with 33 age-matched control (AMC) children. DS serum immunoglobulin levels were compared with 962 non-DS children with recurrent infections. The results were combined with clinical data. Transitional and naive B lymphocytes are profoundly decreased in the children with DS. This could be caused by an intrinsic B-lymphocyte defect resulting in (partial) failure of B-lymphocyte generation, decreased antigen-induced proliferation and/or increased apoptosis, or by decreased proliferation due to deficient T-lymphocyte help, or a combination of these. The decreased CD27, CD21, and CD23 cells are reminiscent of common variable immunodeficiency and suggestive of disturbed peripheral B-lymphocyte maturation. Immunoglobulin levels in DS are abnormal-as has been described before-and different from non-DS children with recurrent infections. We conclude that the humoral immune system is abnormal in DS, but could not find a relation between B-lymphocyte subsets, immunoglobulins and clinical features of the children with DS in our cohort, nor could we answer the question whether DS lymphocytes are truly intrinsically deficient, or could all findings be explained by deficient T-lymphocyte help.

Both normal memory counts and decreased naive cells favor intrinsic defect over early senescence of Down syndrome T lymphocytes.

Because of their increased malignancies, autoimmune diseases, and infections, patients with Down syndrome (DS) show features of immunodeficiency. The DS thymus and T lymphocyte subsets have indeed proven to be different, and this has been interpreted as precocious aging. Our study on T lymphocyte subpopulations in DS shows that the normal expansion of naive helper (CD4CD45RA) and cytotoxic (CD8CD45RACD27) T lymphocytes is lacking in the first years of life; this is more logically explainable with an intrinsic T lymphocyte defect. Furthermore, memory cell numbers are not different from age-matched controls (AMC), which does not support the hypothesis of precocious aging. Although the absolute numbers of T lymphocyte subpopulations approach AMC levels toward adulthood, the persistent clinical problems suggest that these cells may not function optimally. However, the clinical picture does not fit severe T lymphocyte deficiency. The latter concept is also supported by our finding that cytomegalovirus (CMV)-seropositive DS children show similar numbers of terminally differentiated cytotoxic T lymphocytes when compared with healthy children, not increased numbers as are seen in immunocompromised hosts.

Intrinsic abnormalities of lymphocyte counts in children with down syndrome.

OBJECTIVE: Down syndrome (DS) is associated with an increased frequency of infections, hematologic malignancies, and autoimmune diseases, suggesting that immunodeficiency is an integral part of DS that contributes significantly to the observed increased morbidity and mortality. We determined the absolute counts of the main lymphocyte populations in a large group of DS children to gain further insight into this immunodeficiency. STUDY DESIGN: In a large group of children with DS (n = 96), the absolute numbers of the main lymphocyte subpopulations were determined with 3-color immunophenotyping using the lysed whole-blood method. The results were compared with previously published data in healthy children without DS. RESULTS: In healthy children with DS, the primary expansion of T and B lymphocytes seen in healthy children without DS in the first years of life was severely abrogated. The T- lymphocyte subpopulation counts gradually reached more normal levels with time, whereas the B- lymphocyte population remained severely decreased, with 88% of values falling below the 10th percentile and 61% below the 5th percentile of normal. CONCLUSIONS: The diminished expansion of T and B lymphocytes strongly suggests that a disturbance in the adaptive immune system is intrinsically present in DS and is not a reflection of precocious aging. Thymic alterations have been described in DS that could explain the decreased numbers of T lymphocytes, but not the striking B lymphocytopenia, seen in these children.