Chemoluminescence Immunoassay Research Articles

The Role of 25-Hydroxy Vitamin D and Cartilage Oligomeric Matrix Protein (COMP) Serum in Knee Osteoarthritis

Background. Osteoarthritis (OA) is a prevalent disease of the musculoskeletal system that affects over 528 million individuals worldwide, with 73% of those diagnosed being aged 55 or older. The knee joint is most commonly affected, along with the hip and hand joints. Vitamin D is essential for maintaining bone and cartilage metabolism. In chondrocytes, the presence of the vitamin D receptor (VDR) regulates the expression of metalloproteinase. The purpose of this investigation is to examine the correlation between vitamin D (25(OH)D) and cartilage oligomeric matrix protein (COMP) serum in knee osteoarthritis. Methods. The cross-sectional study was conducted by the Rheumatology Division at RSMH Palembang. Patients diagnosed with knee osteoarthritis based on the 1990 ACR criteria were included. The chemoluminescent immunoassay and the Human Cartilage Oligometric Matrix Protein ELISA Reagent were used to measure the levels of vitamin D (25(OH)D) and COMP serum. Results. Thirty participants took part in this study from July to December 2022. According to this study, 33% had insufficiency and 67% had a deficiency of vitamin D. Spearman's Rho test shows that there is a strong negative correlation between vitamin D (25(OH)D) and COMP serum. Conclusion. Vitamin D (25(OH)D) and COMP serum levels were significantly correlated in knee osteoathritis.

The anticancer activity of bovine lactoferrin is reduced by deglycosylation and it follows a different pathway in cervix and colon cancer cells.

Bovine lactoferrin (bLF) is a glycosylated protein with purported beneficial properties. The aim of this work was to determine the role of bLF glycosylation in the adhesion, internalization, and growth inhibition of cancer cells. The viability of cervix (HeLa) and colon (Caco-2) cancer cells (MTT assay and epifluorescence microscopy) was inhibited by bLF, while deglycosylated bLF (bLFdeg) had no effect. Adhesion to cell surfaces was quantified by immunofluorescence assay and showed that bLF was able to bind more efficiently to both cell lines than bLFdeg. Microscopic observations indicated that bLF glycosylation favored bLF binding to epithelial cells and that it was endocytosed through caveolin-1-mediated internalization. In addition, the mechanism of action of bLF on cancer cell proliferation was investigated by determining the amount of phosphorylated intermediates of signaling pathways such as epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) and protein kinase B (known as Akt). Chemoluminescence immunoassay of phosphorylated intermediates showed that bLF inhibited Akt phosphorylation, consistent with its growth inhibiting activity. This assay also indicated that the bLF receptor/signaling pathways may be different in the two cell lines, Caco-2 and HeLa. This work confirmed the effect of glycosylated bLF in inhibiting cancer cell growth and that glycosylation is required for optimal surface adhesion, internalization, and inhibition of the ERK/Akt pathway of cell proliferation through glycosylated cell surface receptors.

Vitamin D and other indicators of calcium-phosphorus metabolism as possible predictors of Parkinson's disease

To study a role of vitamin D and other indicators of calcium-phosphorus metabolism as possible predictors of Parkinson's disease (PD). The main group consisted of 138 patients with PD, the control group included 79 patients without PD. Serum levels of 25-hydroxyvitamin D (25[OH]D) were determined by chemo-luminescence immunoassay. Additionally, the following biochemical markers were evaluated: parathyroid hormone, calcitonin, thyroid-stimulating hormone, thyroxine, alkaline phosphatase, inorganic phosphorus, total calcium, ionized calcium, total protein. In addition, densitometry of the spine (1-4 lumbar vertebrae), proximal femurs, and the middle third of the radius was performed. The relationship between the level of vitamin D in blood serum and clinical data was evaluated using correlation analysis. Regression analysis revealed a statistically significant contribution of the levels of parathyroid hormone, vitamin D, alkaline phosphatase and the T-value of the bone density of the neck of the right hip (T-score NRH) to the probability of PD. In the main group, bone mineral density was significantly different between the groups (p=0.028). Also, there was a high incidence of osteopenia (64%) and osteoporosis (73%). Based on the obtained regression equation, the probability of having PD is p=1/(1+exp2.673-0.007x-0.052y-0.037z-0.012k), where «x» is the parathyroid hormone level, «y» is T-score NRH, «z» is the vitamin D level, «k» is the activity of alkaline phosphatase, exp is the exponent. Vitamin D levels, alkaline phosphatase activity, and T-score NRH have a statistically significant effect on the likelihood of developing PD. With a decrease in the above indicators relative to normal values, the likelihood of having PD increases.

Evaluation of a Novel CLIA Monotest Assay for the Detection of Anti-Hepatitis E Virus-IgG and IgM: A Retrospective Comparison with a Line Blot and an ELISA.

Despite the increasing relevance of Hepatitis E, an emerging disease endemic in developing and with increasing numbers of sporadic cases in industrialized countries, commercial tests are mainly based on batch oriented serological assays. In this retrospective study, we compared a line immunoassay (LIA; recomLine HEV, Mikrogen) and an ELISA (EIA; Anti-Hepatitis E Virus ELISA, Euroimmun) with a novel chemoluminescence immunoassay in a monotest format (CLIA; Hepatitis E VirClia, Vircell). Twenty sera of PCR proven cases of hepatitis E and 68 blood samples serologically pre-characterized were included. Applying the WHO reference standard, the CLIA demonstrated the highest analytical sensitivity for IgG and IgM. The combinations of CLIA/EIA (IgG and IgM) and CLIA/LIA (IgG) measurements showed substantial correlation. Compared to overall antibody detection (seropositivity in ≥2 assays), CLIA correlation was excellent, outperforming LIA (IgM) and EIA (IgG and IgM). Minor IgM cross reactivity in samples of patients with acute EBV infection was observed in all three assays. The CLIA showed good performance in diagnostic samples compared to established LIA and EIA assays. Due to its ready-to-use monotest format, the CLIA allows simple, time- and cost-effective handling of single samples. These qualities make the assay suitable for diagnostics, especially in the emergency setting and for low-throughput laboratories.

Association between testosterone level and coronary artery disease

Background: With increasing age, men experience a gradual decline in testosterone levels. It had long been considered that male sex is a strong risk factor for coronary artery disease (CAD). Aim and Objective: The aim of the study was to evaluate the difference between the Plasma Sex Hormone and the degree of Coronary Artery Stenosis in male patients undergoing Coronary Angiography and in matched controls. Materials and Methods: This study was done at the Department of Physiology, Thanjavur Medical College Tamil Nadu. Participants (n = 80) were recruited from the population in and around Thanjavur. The Institute Ethical Committee approval was obtained for this study. Subjects who met inclusion and exclusion criteria were included in this study as control group: Group A (n = 40) and study group: Group B (n = 40). Informed, written consent was taken from both groups. We studied the Plasma Sex Hormone parameters such as Serum Testosterone level (ng/dL), follicle-stimulating hormone level (mIU/ml), and luteinizing hormone level (mIU/ml) were analyzed by Chemo Luminescence Immunoassay. Before the hormonal assay, the angiogram report was obtained from the cardiologist. Results: The mean testosterone level was 6.837 ± 2.330 for Group A and the mean testosterone level for Group B was 4.967 ± 2.734, for which P = 0.001 (

Clinical evaluation of thrombus molecular markers in patients with malignant tumor

Objective To explore the early diagnostic value of thrombus molecular markers in thrombosis ofpatients with malignant tumors and to evaluate their risk factors. Methods Diagnostic research.A total of 1366 patients (including lung cancer, breast cancer and colorectal cancer,) were randomly selected in the Red Flag Hospital of Mudanjiang Medical College and Mudanjiang Cancer Hospitalfrom September 2009 to February 1919. Among them, 562 were males and 804 were females with average age (59.45±15.10) years old. The control group consisted of 70healthy donors (35 males and 35 females, with an average age of (49.60±19.12) years old), including 69 cases of venous thrombosis (thrombotic group, 32 males and37 females, with an average age of (61.20±15.71) years old).Chemoluminescent enzyme immunoassay was used to detect thromboregulatory proteins(TM), thrombin-antithrombin complexes(TAT), tissue plasminogen activators/inhibitors -1 complexes(t-PAIC), plasminase-anti-fibrinolysis complexes(PIC) in venous plasma. According to the sensitivity and specificity of each marker, the receiver′s work characteristic curve was drawn to evaluate its diagnostic performance. Cox regression analysis was used for single-factor and multi-factor risk analysis. Results The incidence of venous thromboembolism(VTE) in patients with different types of malignant tumors was statistically significant, with lung cancer being the highest, followed by colorectal cancer and breast cancer(P<0.05). The levels of TM, TAT, t-PAIC and PIC were significantly higher in the lung, breast and colorectal thrombosis group than in the control group. The differences were statistically significant(all P<0.05). The optimal cut-off level for TM is 10.57 IU/ml(sensitivity 50.30%, specificity 75.50%, AUC=0.671), and the optimal cut-off level for TAT is 4.16 ng/ml(sensitivity is 80.30%, specificity is 62.80%, AUC=0.757).The optimal truncation level for t-PAIC is 11.44 ng/ml(sensitivity 52.50%, specificity 84.00%, AUC=0.682), and the optimal truncation level for PIC is 1.18μg/ml(sensitivity 67.20%, specificity is 79.50%, AUC=0.790). The combined detection of the four molecular markers has the best sensitivity and diagnostic performance(86.90%, AUC=0.807). Age, stage, metastasis, surgery, tumor diameter, and PIC levels are independent factors that affect the occurrence of VTE in malignant tumors (all P<0.05). Conclusions Different types of malignant tumors have different rates of thrombosis. The combined detection ofTM, TAT, t-PAIC and PIC have the best diagnostic performance, and can be used as a new early diagnosis method for VTE in malignant tumors. Age, stage, metastasis, surgery, and tumor diameter are risk factors for VTE in malignant tumors. PIC levels can be used as a reliable markerfor the risk of VTE in patients with malignant tumors within 6 months. Key words: Neoplasms; Venous thromboembolism; Biomarkers

3,5-T2-A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action.

Over the last decades, thyroid hormone metabolites (THMs) received marked attention as it has been demonstrated that they are bioactive compounds. Their concentrations were determined by immunoassay or mass-spectrometry methods. Among those metabolites, 3,5-diiodothyronine (3,5-T2), occurs at low nanomolar concentrations in human serum, but might reach tissue concentrations similar to those of T4 and T3, at least based on data from rodent models. However, the immunoassay-based measurements in human sera revealed remarkable variations depending on antibodies used in the assays and thus need to be interpreted with caution. In clinical experimental approaches in euthyroid volunteers and hypothyroid patients using the immunoassay as the analytical tool no evidence of formation of 3,5-T2 from its putative precursors T4 or T3 was found, nor was any support found for the assumption that 3,5-T2 might represent a direct precursor for serum 3-T1-AM generated by combined deiodination and decarboxylation from 3,5-T2, as previously documented for mouse intestinal mucosa. We hypothesized that lowered endogenous production of 3,5-T2 in patients requiring T4 replacement therapy after thyroidectomy or for treatment of autoimmune thyroid disease, compared to production of 3,5-T2 in individuals with intact thyroid glands might contribute to the discontent seen in a subset of patients with this therapeutic regimen. So far, our observations do not support this assumption. However, the unexpected association between high serum 3,5-T2 and elevated urinary concentrations of metabolites related to coffee consumption requires further studies for an explanation. Elevated 3,5-T2 serum concentrations were found in several situations including impaired renal function, chronic dialysis, sepsis, non-survival in the ICU as well as post-operative atrial fibrillation (POAF) in studies using a monoclonal antibody-based chemoluminescence immunoassay. Pilot analysis of human sera using LC-linear-ion-trap-mass-spectrometry yielded 3,5-T2 concentrations below the limit of quantification in the majority of cases, thus the divergent results of both methods need to be reconciliated by further studies. Although positive anti-steatotic effects have been observed in rodent models, use of 3,5-T2 as a muscle anabolic, slimming or fitness drug, easily obtained without medical prescription, must be advised against, considering its potency in suppressing the HPT axis and causing adverse cardiac side effects. 3,5-T2 escapes regular detection by commercially available clinical routine assays used for thyroid function tests, which may be seriously disrupted in individuals self-administering 3,5-T2 obtained over-the counter or from other sources.

Cardiac Surgery Successfully Managed With Cangrelor in a Patient With Persistent Anti-PF4/Heparin Antibodies 8 Years After Heparin-Induced Thrombocytopenia

A 66-YEAR-OLD female requiring cardiac surgery had persisting anti-platelet factor 4 (PF4)/heparin antibodies (HIT-abs) 8 years after heparin-induced thrombocytopenia (HIT). In 2010, she developed thrombotic thrombocytopenic purpura (TTP) (ADAMTS-13 <5%, inhibitor at 1.0 BU/mL), which was treated successfully with corticotherapy, plasmapheresis, and intravenous heparin. While taking heparin, she developed HIT, as evidenced by a positive functional test. Her platelet count fully resolved without thrombotic complications with danaparoid treatment. In 2018, the preoperative titer of HIT-abs was still 0.38 U/mL by chemoluminescent immunoassay (CLIA), and positive by particle-gel agglutination immunoassay (PaGIA) with a titer of 2 and was strongly positive on an enzyme-linked immunosorbent assay (ELISA). The authors of the case report chose to use cangrelor combined with heparin during cardiopulmonary bypass (CPB). Cangrelor was used without increased postoperative bleeding or thrombotic complications. Postoperatively she exhibited a huge rise in HIT-abs (14.22 U/mL on postoperative day 11) with a positive functional assay. There was no recurrence of HIT, however. This case illustrates the importance of excluding the presence of persisting HIT-abs before CPB and ensuring close medical follow-up after even a single exposure to heparin.

PF326 VITAMIN D INSUFFICIENCY AND PROGNOSIS IN LYMPHOMA

Background:Vitamin D deficiency has been recently associated, as an independent risk factor, with lower rates of progression‐free survival (PFS) and overall survival (OS) in patients with Hodgkin (HL) and non‐Hodgkin's lymphoma (NHL).Aims:To investigated the impact of vitamin D status on the outcome of lymphoma patients in two Institutions.Methods:From 2015 to 2018, we tested circulating 25‐hydroxyvitamin D3 (25[OH]D3) levels in a prospective cohort of 135 newly diagnosed patients with NHL and HL in two Institutions in Spain. 25[OH]D3 levels were measured by chemoluminescent immunoassay. Determinations were based on guidelines from the Food and Nutrition Board of the Institute of Medicine. According to current guidelines, we considered the following thresholds: vitamin D deficient (&lt;10 ng/ml), vitamin D insufficiency (10 to 30 ng/ml) and normal vitamin D level (&gt;30 ng/ml). Patients with &lt;20 ng/ml from one Institution had vitamin D substitution. OS and PFS were calculated using Kaplan‐Meier estimators, comparison between categories performed by log‐rank test and Cox PH regression, and the effect of covariates reported with 95% confidence interval (95 CI).Results:135 newly diagnosed patients with NHL and HL were included. Median age at diagnosis was 67 years (range, 20 to 91 years). NHL subtypes: 47% diffuse large b cell lymphoma (DBCL), 30% Indolent lymphoma, 5% mantle cell lymphoma, 6% Tcell lymphoma, 10% Hodgkin lymphoma and 3% others NHL 34% had advanced stage and and 39% of NHL patient had ≥ 3score of International Pronostic Index (IPI).55% of the patients were diagnosed in spring or summer. Median 25(OH)D levels at diagnosis were 18 ng/ml. 11% of patients had normal 25(OH)D levels, 77% had insufficient 25(OH)D levels and 12% were deficient at lymphoma diagnosis. Median follow‐up was 12 months and 22 deaths were observed. The three groups of vitamin D levels were associated with OS, but this difference was not statistically significant (Figure 1).In the DLBCL subset, we inspected in a Cox Model the effect of Vitamin D levels for OS/PFS, adjusting for IPI Score and vitamin D substitution. Vitamin D levels did not achieve statistical significance for PFS (hazard ratio [HR], 1.02; 95% CI, 0.99 to 1.04) and OS (HR: 0.99; 95% CI, 0.94 to 1.06). Remarkably, there was a tendency towards a better statistically significant outcome in patients supplemented (HR for PFS: 0.36; 95% CI, 0.10 to 1.29; p = 0.10).Summary/Conclusion:Most of our patients present with low levels of vitamin D[JPD1]. In our limited series, with the current follow‐up time, baseline vitamin D values did not significantly impact PFS and OS. Further studies are needed to confirm our data.image

No improvement in vitamin D status in German infants and adolescents between 2009 and 2014 despite public recommendations to increase vitamin D intake in 2012

PurposeVitamin D is a key component for the growth and development of children and adolescents, influencing a multitude of functions. Worldwide epidemiological studies have shown that minimum vitamin D blood levels of ≥ 20.0 ng/ml, often defined as vitamin D sufficiency by international and national nutrition and pediatric organizations, are often not met in practice. In 2012 the D–A–CH (Germany, Austria, Switzerland) nutrition societies increased their vitamin D intake recommendations fourfold from 200 IU (5 µg) to 800 IU (20 µg) per day. The outcome of this study will contribute to answering the question as to whether the new recommendations for increased vitamin D intake improve the highly prevalent vitamin D deficiency status in German children and adolescents.MethodsFor this 6-year study (January 2009–December 2014) carried out in Mülheim an der Ruhr, Germany, healthy children and adolescents (n = 1929, age range 1–17 years, median age 11.0 years, 46.9% female) consulting a pediatric group practice (KIDS4.0) were recruited. Serum 25(OH)D determinations were performed using a competitive chemoluminescence immunoassay (CLIA, DiaSorin).ResultsThe median serum vitamin D values for each year from 2009 to 2014 were 18.4, 13.0, 20.8, 16.4, 19.4 and 14.9 ng/ml. The summarized median 25(OH)D serum concentrations between the two time periods 2009–2012 and 2013–2014 after increasing recommendations for vitamin D intake did not show a significant difference (17.0 versus 16.8 ng/ml).ConclusionsThe increased D–A–CH recommendations for vitamin D intake had no influence on vitamin D levels in children and adolescents. The prevalence of vitamin D deficiency has not changed compared to previous studies.

Clinical monitoring of serum sex hormones during normal menstrual follicle growth cycle

Objective To study the changes of six sex hormones corresponding to the follicle growth during the normal menstrual cycle of Chinese women. Methods Thirty Chinese women with regular menstrual period and average age of (28.8±3.2) years were selected for the study by Peking Union Medical College Hospital in September, 2010. Growth of follicles was monitored by using transvaginal sonography. Six sex hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), testosterone (T), and prolactin (PRL) were measured by chemoluminescence immunoassay every day during a menstrual cycle. Nonparametric statistical analysis was used. Results Menstrual cycle of all the patients was in the range of 25 to 39 d, with mean of (29.5 ± 3.1) d. Length of follicular phase and luteal phase was 15.3 and 14.4 d, respectively.Number of days from antral follicle to emergence of dominant follicle, and from the latter to ovulation, was 6.2 and 8.9 d, respectively. Average diameter of preovulatory follicle was 19.33 mm. Both FSH and LH reached peak on the day before ovulation. P started to increase before ovulation and remained at a high plateau from 6th to 9th day after ovulation. Both PRL and T reached peak after ovulation, near the end of a menstrual cycle. Conclusions A small rise of LH and P emerges just 1 to 2 d before ovulation, indicating the maturing of follicle. PRL and T shows cyclic changes as follicle grows. Therefore, PRL and T levels should be measured in the early follicle phases in the clinical practice so that leading the impact of menstrual cycle minimal.(Chin J Lab Med, 2017, 40: 169-173) Key words: Menstrual cycle; Ovarian follicle; Gonadal steroid hormones; Luminescent measurements; Immunoassay

Diagnostic of Helicobacter pylori infection.

There is progress in endoscopy techniques. While it is not yet possible to detect Helicobacter pylori directly in the stomach, it becomes easier to detect the mucosal changes induced by the bacteria. Some small changes can also increase the sensitivity of the invasive tests, for example culture or histology, but the wide use of proton-pump inhibitors has a negative impact on these tests. Only molecular methods are able to detect a limited load of bacteria, especially by using real-time PCR but also with new methods, for example dual-priming oligonucleotide-based PCR, loop-medicated isothermal amplification, droplet-digital PCR or a multiple genetic analysis system. Among the noninvasive tests, urea breath test remains a test of major interest, while there are attempts to develop an ammonia breath test and other nanosensor devices. A new antigen stool test, a chemoluminescence immunoassay using the LIAISON apparatus has also been tested for the first time with success. Despite its limitations, serology remains the most popular test to detect H.pylori antibodies. It also allows pepsinogen dosage which is of interest for detecting atrophy.

EPSTEIN-BARR VIRUS AND CYTOMEGALOVIRUS: IS THEIR ROLE IN PEMPHIGUS REALLY INCIDENTAL? A PRELIMINARY REPORT

Актуальность. К одним из наиболее распространенных среди популяции всего земного шара инфекций, ассоциированных с аутоиммунными процессами, относят инфекции, вызванные вирусом Эпштейна – Барр (ВЭБ) и цитомегаловирусом (ЦМВ). Однако противоречивые данные исследований по роли герпесвирусных инфекций в развитии аутоиммунной пузырчатки не позволяют уверенно рассматривать данные вирусы как триггерный фактор в возникновении и течении этого буллезного дерматоза. Цель – изучение наличия специфических IgM-, IgG-антител к группе герпесвирусных инфекций у больных аутоиммунной пузырчаткой. Материал и методы. Исследованы образцы сыворотки крови 15 больных аутоиммунной пузырчаткой методом хемилюминесцентного иммуноанализа. Результаты. В сыворотке крови у 14 (93,3%) из 15 больных аутоиммунной пузырчаткой одновременно выявлены специфические IgG-антитела к ядерному и капсидному белкам ВЭБ в диапазоне от 30,7 до 600 Ед/мл (медиана 147,5 [102,62; 313,25] Ед/мл) и от 33,5 до 567 Ед/мл (медиана 186 [85,95; 492,5] Ед/мл) соответствен-но. Специфические IgG-антитела к раннему белку ВЭБ были обнаружены только в 6,7% случаев. Специфические IgM-антитела к антигенам оболочки ВЭБ отсутствовали у всех обследованных больных. У всех (100%) больных в сыворотке крови выявлены специфические IgG-антитела к ЦМВ в диапазоне от 64,5 до 138 Ед/мл (медиа-на 103,5 [94,83; 113,75] Ед/мл). В 30% случаев обнаружены специфические IgM-антитела к ЦМВ, их титр варьировал от 11 до 12,3 Ед/мл (медиана 5 [5; 9,5] Ед/мл). Заключение. Pезультаты предварительного исследования показали, что в 93,3% случаев аутоиммунная пузырчатка протекает на фоне хронической инфекции, вызван-ной ВЭБ и ЦМВ. При этом выявленный высокий уровень IgG-антител к антигенам ВЭБ и ЦМВ позволяет говорить о том, что ассоциация этих вирусов с данным буллезным дерматозом не является случайной. Несомненно, это требует продолжения научных исследований, результаты которых позволят сделать более точные выводы о роли ВЭБ и ЦМВ в патогенезе аутоиммунной пузырчатки и сформировать новый взгляд на лечение пациентов, страдающих столь угрожающим для жизни заболеванием.

Vitamin D deficiency impairs rituximab-mediated cellular cytotoxicity and outcome of patients with diffuse large B-cell lymphoma treated with but not without rituximab.

To investigate the impact and mechanisms of vitamin D deficiency (VDD) on the outcome of elderly patients with diffuse large B-cell lymphoma (DLBCL). Three hundred fifty-nine pretreatment 25-hydroxyvitamin D3 (25[OH]D3) serum levels from the RICOVER-60 study (Six Versus Eight Cycles of Biweekly CHOP-14 With or Without Rituximab in Elderly Patients With Aggressive CD20+ B-Cell Lymphomas) and 63 from the RICOVER-noRTh study (an amendment to the RICOVER-60 study in which patients received six cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone administered at an interval of 2 weeks plus two cycles of rituximab [R-CHOP-14], but without radiotherapy) were determined by chemoluminescent immunoassay. Rituximab-mediated cellular cytotoxicity (RMCC) was assessed by lactate dehydrogenase release assay of CD20+ Daudi cells. RICOVER-60 patients with VDD (≤ 8 ng/mL) and vitamin D levels more than 8 ng/mL treated with rituximab had 3-year event-free survival (EFS) of 59% and 79% and 3-year overall survival (OS) of 70% and 82%, respectively. These differences were significant in a multivariable analysis adjusting for International Prognostic Index risk factors with a hazard ratio (HR) of 2.1 (P = .008) for EFS and 1.9 (P = .040) for OS. EFS was not significantly different in patients with vitamin D levels ≤ 8 or more than 8 ng/mL (HR, 1.2; P = .388) treated without rituximab. This was confirmed in an independent validation set of 63 RICOVER-noRTh patients. RMCC increased significantly (P < .001) in seven of seven individuals with VDD after substitution and normalization of their vitamin D levels. VDD is a risk factor for elderly patients with DLBCL treated with R-CHOP. That VDD impairs RMCC and substitution improves RMCC strongly suggests that vitamin D substitution enhances rituximab efficacy, which must be confirmed in appropriately designed prospective trials addressing VDD and substitution not only in DLBCL, but also in malignancies treated with other antibodies, of which the major mechanism of action is antibody-dependent cellular cytotoxicity (eg, trastuzumab in breast cancer and cetuximab in colorectal cancer).

25-OH-Vitamin-D Deficiency Impairs Rituximab-Mediated Cellular Cytotoxicity and Is Associated With An Inferior Outcome Of Elderly DLBCL Patients Treated With Rituximab

BackgroundTo investigate the impact and underlying mechanisms of vitamin-D-deficiency (VDD) on outcome of elderly (61 to 80 year-old) DLBCL patients. MethodsPretreatment 25-OH-vitamin-D serum levels from 359 patients treated in the prospective multicenter RICOVER-60 trial with 6 or 8 cycles of CHOP-14 with and without 8 cycles rituximab and 63 patients in the RICOVER-noRT study treated with 6xCHOP-14 + 8xR were determined determined by LIASION®, a commercially available chemoluminescent immunoassay. ResultsRICOVER-60 patients with VDD (defined as serum levels ≤8 ng/m l) and treated with rituximab had a 3-year event-free survival of 59% compared to 79% in patients with >8 ng/ml; 3-year overall survival was 70% and 82%, respectively. These differences were significant in a multivariable analysis adjusting for IPI risk factors with a hazard ratio of 2.1 [p=0.008] for event-free survival and 1.9 [p=0.040] for overall survival. In patients treated without rituximab 3-year EFS was not significantly different in patients with vitamin-D levels ≤8 and >8 ng/ml (HR 1.2; p=0.388). These results were confirmed in an independent validation set of 63 patients treated within the RICOVER-noRT study. Rituximab-mediated cellular toxicity (RMCC) against the CD20+ cell line Daudi as determined by LDH release assay increased significantly (p<0.005) in 5/5 vitamin-D-deficient individuals after vitamin-D substitution and normalization of their vitamin-D levels. ConclusionsVDD is a significant risk factor for elderly DLBCL patients treated with rituximab. Our results show that VDD impairs RMCC and that RMCC can be improved by vitamin-D substitution. This together with the differential effect of VDD in patients treated with and without rituximab suggests that vitamin-D substitution might result in a better outcome of these patients when treated with CHOP plus rituximab. Supported by a grant from Deutsche Krebshilfe. Disclosures:No relevant conflicts of interest to declare.

Nivel sérico de folato y vitamina B12 en adultos mayores chilenos: Resultados de la Encuesta Nacional de Salud ENS 2009-2010

Supraphysiological levels (SFL) of serum folate (SF) derived from flour fortification with folic acid (FA) could be risky among older adults with low vitamin B12 (B12) levels. To describe and analyze SF and B12 levels in older Chileans and to identify risk groups. Participants were 1.043 older people aged ≥ 65 years from the National Health Chilean Health Survey 2009-2010 (ChNHS 2009-10), a multistage stratified random sample, representative of the national population. SF (µg/L) and B12 (pg/ml) were determined in fasting samples by competitive chemoluminescence immunoassay. Mean, deciles and percentiles 5 and 95th were calculated. We defined SF categories: < 4.4 (deficit); 4.41-20 (normal) and SFL: 20.01-25.6; 25.6-29 and > 29 µg/L (80th percentile of the distribution) and vitamin B12 categories: ≤ 200 (deficit); 200.1-299.5 (marginal deficit) and > 299.5 (normal). Prevalence rates, multiple and logistic regression models were used and adjusted by sex and age, educational level and residence area. SF and B12 mean and 95th percentiles were 21.2 ± 0.56/38.6 µg/L and 348.4 ± 7.6/637(pg/ml) respectively. Forty nine percent of participants had SFL. Folate and B12 deficiency were present in 0.3 and 8.1% of participants, respectively. Men had significantly lower prevalence of SFL > 29 µg/L (OR adjusted odds ratio 0.47 95% confidence intervals: 0.26-0.84). B12 showed no significant variation by age and sex. The prevalence of SFL associated with B12 deficiency was 4.1%. No statistically significant association was observed between levels of folate and B12. Folate deficit is almost inexistent, but a significant percentage of participants had SFL suggesting the need for revising the current wheat flour fortification levels.

Differential expression of ISG20 in chronic hepatitis B patients and relation to interferon‐alpha therapy response

The 20 kDa exonuclease encoded by the interferon-stimulated gene, ISG20, can inhibit the replication of hepatitis B virus (HBV), and may represent a clinically useful prognostic marker for response to interferon-alpha (IFN-α) antiviral therapy. The present study was designed to investigate the differential expression patterns of ISG20 in liver biopsy samples from treatment-naive patients with chronic hepatitis B and non-HBV infected controls and to determine the relation between the differential expression and IFN-α treatment outcome (responders vs. non-responders). HBV infection status was determined by measuring levels of hepatitis B surface antigen (HBsAg) by chemoluminescence immunoassay and of HBV DNA by real-time quantitative (q)PCR. ISG20 protein and mRNA expressions were assessed by immunohistochemistry and qPCR, respectively. Chronic hepatitis B responders showed significantly higher levels of ISG20 protein and mRNA expressions than either the chronic hepatitis B non-responders or the controls. Moreover, increased expression of ISG20 in both the nucleus and cytoplasm was correlated with positive response to IFN-α treatment. Thus, active transcription and translation of ISG20 may represent a marker to identify chronic hepatitis B patients likely to respond to IFN-α therapy. Prognostic clinical strategies based upon this marker may include genomic screening methods and immunohistochemical staining of liver biopsies.

Association of 25-OH vitamin D deficiency with worse outcome for elderly patients with aggressive B-cell lymphomas treated with CHOP plus rituximab (R): An analysis of the RICOVER-60 trial of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL).

8569 Background: Vitamin D deficiency was shown to be is associated with a worse outcome in patients with non-Hodgkin's lymphoma (Drake et al., 2010) To study whether this observation could be confirmed in patients with aggressive B-cell lymphomas treated uniformly within a prospective trial, we analyzed 25-OH vitamin D serum levels in patients treated within the RICOVER-60 trial of the DSHNHL. Methods: 25-OH Vitamin D serum levels were determined with a commercial chemoluminescence immunoassay in the serum from elderly patients of the RICOVER-60 trial which compared 6 or 8 cycles of CHOP, both with and without rituximab. Results: 193 of 359 pts (53.8%) had vitamin D deficiency (&lt;10 ng/ml) and 165/359 patients (46.0%) had vitamin D insufficiency (10-30 ng/ml) according to current definitions. When treated with R-CHOP, patients with vitamin D levels ≤8 ng/ml had a 3-year EFS of 59% compared to 79% of patients with vitamin D serum levels &gt;8 ng/ml; the respective figures for 3-year overall survival were 70% and 82%, respectively. In R-CHOP pts these differences were significant in a multivariable analysis adjusting for IPI risk factors with a hazard ratio (HR) of 2.1 (p=0.008) for EFS and a HR of 1.9 (p=0.040) for OS. In pts treated without R effects of vitamin D deficiency were significant only for OS (HR 1.8; p=0.025), but not with respect to EFS (HR 1.2; p=0.388). These results were confirmed in an independent validation set of 63 patients treated within the prospective RICOVER-noRx study. Conclusions: Vitamin D deficiency is a significant risk factor for patients with aggressive B-cell lymphomas treated with R-CHOP. The stronger adverse effect of vitamin D deficiency in patients receiving rituximab suggests that vitamin D deficiency interferes with the R mechanisms of this antibody. A prospective study evaluating the effects of vitamin D substitution on outcome of patients receiving R-CHOP is warranted. Supported by Deutsche Krebshilfe.

Seroprevalence for toxoplasmosis in individuals living in North West Tuscany: access to Toxo-test in central Italy

This survey aimed to estimate the prevalence of anti-Toxoplasma IgG and IgM antibodies in people living in north west Tuscany (central Italy) and to investigate the adherence to antenatal screening programs and access to the Toxo-test as well. Sera from a large sample of individuals suspected to have acute infection or from pregnant women (10,352 subjects) aged between 1 day and over 70 years were analysed for both IgG and IgM anti-Toxoplasma antibodies using an immunoenzymatic method or a chemo-luminescent immunoassay. Overall, the seroprevalence of IgG antibodies was 21.4% (95% CI 20.62-22.20). A positive trend according to age was found, with low positivity observed in younger age groups. Among women of reproductive age the prevalence of IgG antibodies was 19.4% (95% CI 18.64-20.26). The overall IgM seroprevalence was 1.07% (95% CI 0.87-1.27). A low IgM prevalence was also observed in women of reproductive age (0.8%; 95% CI 0.65-1.03). Our study seems to indicate that primary prevention is widespread among women. However, an epidemiological surveillance system for toxoplasmosis should be implemented, to assess the risk of congenital toxoplasmosis and to determine the true burden of disease in adults.

Age-related testosterone decline in a Brazilian cohort of healthy military men

Androgen decline in the aging man has become a topic of increasing clinical relevance worldwide, as the reduction in testosterone levels has been reported to be accompanied by loss of muscle mass, accumulation of central adiposity, impaired mobility and increase risk of bone fractures. Although well-established in studies conducted in developed countries, progressive decline in serum testosterone levels with age has been poorly investigated in Brazil. To determine the pattern of blood testosterone concentrations decline with age in a cohort of Brazilian healthy military men. We retrospectively reviewed data on serum testosterone measurements of healthy individuals that had undergone a routine check-up at the Military Biology Institute. Blood samples were obtained early in the morning, and total testosterone concentration was determined using a commercial chemoluminescent immunoassay. Mean values were analyzed in five age groups: ≤ 40, 41 to 50, 51 to 60, 61 to 70, and > 70 years. Mean total testosterone levels. 1,623 subjects were included in the analysis; mean age was 57 years (24 to 87), and mean testosterone level was 575.5 ng/dL (25.0 to 1308.0 ng/dL). The evaluation of age-related changes in total testosterone levels revealed a progressive reduction in serum levels of this hormone with increasing age. Testosterone levels below 300 ng/dL were reported in 321 participants, a prevalence of nearly 20% in the study population. In agreement with other findings, a reduction of total testosterone levels with age was reported for healthy Brazilian men.