Follow-up Blood Samples Research Articles

Impact of Proton-Pump Inhibitor Use on Gut Microbiota Composition and Other Markers of Health in Older Adults

The composition and diversity of the gut microbiota interact with the host immune system to prevent infection and limit accumulation of pathogen-related toxins. Proton-pump inhibitors (PPIs) may affect the gut microbiota by directly targeting bacterial proton pumps and/or affecting the microenvironment of the flora; however, the extent to which PPIs alter gut microbiota composition and other markers of health is unknown. This was a prospective study of healthy volunteers from San Antonio, TX from June to November 2016. Included subjects were ≥60 years old and had no recent use of medications known to affect the gut microbiota. Participants provided a blood and stool sample at baseline, completed a 14-day course of omeprazole 20mg daily, then provided a follow-up blood and stool sample. Stool 16s rRNA sequences were classified into operational taxonomic units (OTUs) via Mothur’s Bayesian classifier and referenced to the Greengenes database. OTU richness and Shannon diversity were compared between samples using the Wilcoxon signed rank test. Abundance-weighted sample differences were calculated using the Bray-Curtis dissimilarity. PERMANOVA was used to assess the impact of PPI use on β diversity. Systemic inflammatory markers (interleukin [IL]-6, IL-10, and tumor necrosis factor [TNF]-alpha) and insulin-like growth factor (IGF-1) were analyzed using ELISA and compared using the paired t-test. A total of 24 subjects completed the study (mean age 71 years; 63% female). OTU richness (P = 0.32) and Shannon diversity (P = 0.28) were similar between pre- and post-PPI samples. PPI use was associated with significantly lower abundance of Actinobacteria, Lachnospiraceae, Erysipelotrichaceae, and Bifidobacteriaceae. Β diversity was significantly associated with PPI use (P < 0.0001). PPI use was associated with a significant increase in mean IL-10 (5.7 vs. 4.7, P = 0.0385) and decrease in mean IGF-1 (87.5 vs. 94.8, P = 0.0185), while TNF-alpha (1.4 vs. 1.4, P = 0.9180) and IL-6 (4.6 vs. 3.4, P = 0.0808) were statistically similar between groups. Short-term, over-the-counter PPI use was associated with significant changes in gut microbiota composition and other markers of health in older adults. All authors: No reported disclosures.

Assessment of different folic acid supplementation doses for low-birth-weight infants.

The adequacy of 50 mcg folic acid supplementation given to low-birth-weight babies was investigated. The folate levels of the mothers and infants, and breastmilk, and the optimum dose for folic acid supplementation were also investigated. After obtaining blood from 141 low-birth-weight infants on the 1st day of life for serum and red cell folate levels, the infants were randomly allocated into three groups according to the folic acid supplement dose. Forty-six infants were given 25 μg/d folic acid, 39 were given 50 μg/d folic acid, and 44 were given 75 μg/d folic acid. Folic acid could not be given to 12 infants. Follow-up blood samples were obtained at the end of folic acid supplementation. Maternal samples for red cell and serum folate levels and breast milk folate levels were obtained within the first 48 hours and the samples for measuring breastmilk folate level were obtained on the 3rd day postnatally. The feeding modes of the infants, maternal folic acid intake, and details of neonate intensive care unit course were recorded. The mean birth weight and gestational age of the infants were found as 1788.2±478.4 g and 33.5±2.9 weeks, respectively. The mean serum and red cell folate levels on admission were found as 21.2±12.2 ng/mL and 922.7±460.7 ng/mL, respectively. The mean maternal serum and red cell folate levels and the mean breast milk folate levels were found as 12.3±7.5 ng/mL, 845.5±301.4 ng/mL, and 30.6±33.0 ng/m, respectively. The breast milk folate levels of mothers who were supplemented with folic acid during pregnancy were significantly higher compared with mothers who were not supplemented with folic acid (p<0.001). Infants who were supplemented with folic acid had higher follow-up serum folate levels compared with the basal level in all groups, but there was no statistically significant difference between the groups. This study showed that the folic acid doses of 25, 50, and 75 μcg/d affected serum folate levels similarly. We can conclude that the dose of 25 μcg/d is adequate for low-birth-weight infants.

The effect of iron versus iron plus zinc supplementation in children with malaria

Introduction Little is known about the potential interaction of ironand zinc given to increase hemoglobin and serum ferritin in chil-dren with malaria.Objective To study the effect of iron compared with a combination ofiron and zinc supplementation on children with falciparum malaria.Method Children with positive Plasmodium falciparum (n=86) wererandomly assigned to a daily supplementation of 6 mg iron/kg perday plus placebo or plus 10 mg zinc per day for 30 days. All childrenwere treated with the same regimen for the treatment of P. falciparum.Venous blood samples were collected at the start and end of thestudy. After 30 days of supplementation, the baseline and follow-upblood samples were analyzed.Results The increase of hemoglobin concentration in the ironplus placebo group was 0.58 g/dl, while in the iron plus zinc groupwas 0.09 g/dl (P&lt;0.05). Serum ferritin concentration was high inboth groups before trial, yet there was no significant differenceafter iron supplementation.Conclusions Iron supplementation showed significant increasein hemoglobin concentration in children with positive P. falciparumtreated with the same regimen of treatment. Supplementation ofiron alone as well as iron plus zinc had been proven ineffective toiincrease serum ferritin in children with malaria.

Sofosbuvir/Ledipasvir Performance in Genotype 3 HCV Infection: Real-Life Evidence of Overcoming a Difficult Patient Subgroup

Introduction: The combination of sofosbuvir (SOF) and ribavirin (RBV) for treatment of HCV genotype 3 infection is suboptimal, in particular for patients with cirrhosis who have previously failed IFN and ribavirin. Pending further evidence, the combination of sofosbuvir plus ledipasvir (SOF/LDV) is not recommended in patients infected with HCV genotype 3. We aimed to compare the efficacy of SOF+RBV to SOF/LDV + RBV in a cohort of patients with HCV genotype 3 infection. Methods: Genotype 3 HCV infected patients were randomly assigned for treatment with SOF 400mg plus LDV 90mg plus RBV or with SOF 400mg plus RBV, both during 24 weeks. Viremia was sequentially evaluated at baseline, week 4, 12 and week 24, all with follow-up blood sampling at week 12 post treatment. Results: A total of 71 patients (mean age 52.5±8.5) were treated: 82% males, 44% cirrhotic, 44% treatment experienced. Thirty-four (48%) patients were treated with SOF+RBV and the remaining 52% with SOF/LDV+RBV. There were no differences between both treatment groups, namely regarding degree of fibrosis, viral load and prior treatment response. Among patients treated with SOF+RBV, treatment response at week 4, 12 and 24 was 87%, 92% and 100%, respectively, and 82% of the patients achieved sustained virologic response at week 12-post treatment. Among patients treated with SOF/LDV+RBV, treatment response at week 4, 12 and 24 was 81%, 100% and 100%, respectively and 97% of the patients achieved sustained virologic response at week 12 post treatment. Sustained virologic response was higher in the group treated with SOF/LDV+RBV compared to the group treated with SOF+RBV (97% versus 82%, p=0.041). Baseline viral load, previous treatment response and degree of fibrosis did not influence treatment response for both treatment groups. Conclusion: In this real life cohort of HCV genotype 3infected patients, treatment with SOF/LDV+RBV achieved a higher sustained virologic response compared to treatment with SOF+RBV.

75P - PD-L1 expression on circulating CD45(-) cells is an independent prognostic factor for overall survival (OS) in patients (Pts) across all stages of treatment-naïve lung cancer in a prospective, multicenter study

Biopsies are currently utilized for profiling (histologic, molecular) of lung cancer prior to treatment. Profiling tumors from circulating markers could avoid invasive procedures and enable more frequent, kinetic disease monitoring. PD-L1 expression on lung cancer biopsy correlates with efficacy of immune checkpoint inhibitors. Using the Epic Sciences PD-L1 assay, we sought to correlate patient outcome with PD-L1(+) circulating CD45(-) cells, prior to its evaluation as a predictive biomarker for immune checkpoint inhibitors. Peripheral blood was collected from 124 pts with diverse staging and histology at three clinical sites prior to diagnostic biopsy, with some having follow-up draws. All nucleated cells were plated onto glass slides and circulating cells of interest identified by immunofluorescence and morphological features. Prognostic capacity of PD-L1(+) cells (CK + /-, CD45-, PD-L1 + , malignant nuclear morphology) were assessed with Kaplan-Meier and Cox Proportional Hazard (PH) models. Results:Tabled 1AJCC Stagen = 124 Baselinen = 27 Follow-UpI537II184III308IV228 Open table in a new tab When detected, PD-L1 subcellular localization was primarily membranous. Pts with >1 PD-L1(+) cell/mL in baseline samples had worse overall survival (OS) (n = 22/124, mOS: 17.2 months vs. not reached, HR = 3.22, p = 0.0015) and follow-up (n = 5/22 mOS = 2.1 months vs. not reached, HR = 4.25, p = 0.0302). High baseline PD-L1(+) cell burden was additive and independent to AJCC staging in Cox PH models (HR = 2.32, p = 0.0447). Single-cell sequencing is underway. In a multicenter cohort, circulating PD-L1(+)/CD45(-) cell burden predicted worse OS in pre-biopsy and follow-up lung cancer blood samples. This warrants prospective investigation as a predictive biomarker to PD-1 axis immune checkpoint inhibitors.

Prognostic impact of circulating tumor cell apoptosis and clusters in serial blood samples from patients with metastatic breast cancer in a prospective observational cohort.

BackgroundPresence of circulating tumor cells (CTCs) is a validated prognostic marker in metastatic breast cancer. Additional prognostic information may be obtained by morphologic characterization of CTCs. We explored whether apoptotic CTCs, CTC clusters and leukocytes attached to CTCs are associated with breast cancer subtype and prognosis at base-line (BL) and in follow-up (FU) blood samples in patients with metastatic breast cancer scheduled for first-line systemic treatment.MethodsPatients with a first metastatic breast cancer event were enrolled in a prospective observational study prior to therapy initiation and the CellSearch system (Janssen Diagnostics) was used for CTC enumeration and characterization. We enrolled patients (N = 52) with ≥5 CTC/7.5 ml blood at BL (median 45, range 5–668) and followed them with blood sampling for 6 months during therapy. CTCs were evaluated for apoptotic changes, CTC clusters (≥3 nuclei), and leukocytes associated with CTC (WBC-CTC, ≥1 CTC + ≥1 leukocytes) at all time-points by visual examination of the galleries generated by the CellTracks Analyzer.ResultsAt BL, patients with triple-negative and HER2-positive breast cancer had blood CTC clusters present more frequently than patients with hormone receptor-positive cancer (P = 0.010). No morphologic characteristics were associated with prognosis at BL, whereas patients with apoptotic CTCs or clusters in FU samples had worse prognosis compared to patients without these characteristics with respect to progression-free (PFS) and overall survival (OS) (log-rank test: P = 0.0012 or lower). Patients with apoptotic or clustered CTCs at any time-point had impaired prognosis in multivariable analyses adjusting for number of CTCs and other prognostic factors (apoptosis: HROS = 25, P < 0.001; cluster: HROS = 7.0, P = 0.006). The presence of WBC-CTCs was significantly associated with an inferior prognosis in terms of OS at 6 months in multivariable analysis.ConclusionsPatients with a continuous presence of apoptotic or clustered CTCs in FU samples after systemic therapy initiation had worse prognosis than patients without these CTC characteristics. In patients with ≥5 CTC/7.5 ml blood at BL, morphologic characterization of persistent CTCs could be an important prognostic marker during treatment, in addition to CTC enumeration alone.Clinical Trials (NCT01322893), registration date 21 March 2011Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2406-y) contains supplementary material, which is available to authorized users.

Evaluation of parameters obtained from the Sysmex XN-2000 for predicting the recovery of the absolute neutrophil count and platelets after hematopoietic stem cell transplantation.

We analyzed abilities of parameters from Sysmex XN-2000 (Sysmex, Kobe, Japan) to predict absolute neutrophil count (ANC) and platelet recovery after hematopoietic stem cell transplantation (HSCT) in patients with hematologic malignancies. We prospectively analyzed 911 follow-up peripheral blood samples from 44 HSCT-performed patients and evaluated the performances of the following parameters: WBC, immature granulocyte (IG), hematopoietic stem and progenitor cells (HPC), immature reticulocyte fraction (IRF), immature platelet fraction (IPF), platelet distribution width (PDW), mean platelet volume (MPV), and platelet larger cell ratio (P-LCR). When compared to four other parameters, the identification of initiation in IG (%)/HPC (%) increase enabled earlier prediction of ANC recovery to >500/μL and >1000/μL with more time benefit of 3.5-6.5 days/2.0-5.0 days and 3.0-6.0 days/2.0-5.0 days, respectively. When compared to IPF (%), the identification of initiation in PDW, MPV, and P-LCR (%) increase enabled earlier prediction of platelet recovery to >20 000/μL and >50 000/μL with more time benefit of 2.5-3.5 days and 2.0-3.0 days, respectively. However, the standard deviation of time benefit obtained from IG (%)/HPC (%)/PDW/MPV/P-LCR (%) was consistently large (3.0-4.3 days). There is a systematic pattern where a rise in most of the studied parameters can be observed in most patients before ANC/platelet recovery. However, the interindividual variation between the time of rise of these parameters and ANC/platelet recovery is large, and therefore, using these parameters to predict recovery in the individual patient is probably not meaningful in the clinical setting.

A prospective study on the incidence of Borrelia burgdorferi sensu lato infection after a tick bite in Sweden and on the Åland Islands, Finland (2008–2009)

Lyme borreliosis (LB) is a common and increasing tick-borne disease in Europe. The risk of acquiring a Borrelia infection after a tick bite is not fully known. Therefore, we investigated the incidence of Borrelia infection after a bite by a Borrelia-infected tick and if the Borrelia load and/or the duration of tick-feeding influenced the risk of infection. During 2008–2009, ticks and blood samples were collected from 1546 tick-bitten persons from Sweden and the Åland Islands, Finland. Follow-up blood samples were taken 3 months after the tick bite. The duration of tick feeding was microscopically estimated and Borrelia was detected and quantified in ticks by real-time PCR. Anti-Borrelia antibodies were detected in sera using ELISA tests and immunoblot.Five percent (78/1546) of the study participants developed Borrelia infection (LB diagnosis and/or seroconversion) after a tick bite (45% bitten by Borrelia-infected ticks and 55% bitten by uninfected ticks). Of these, 33 developed LB (whereof 9 also seroconverted) while 45 participants seroconverted only. Experience of non-specific symptoms was more frequently reported by Borrelia-infected participants compared to uninfected participants. All who seroconverted removed “their” ticks significantly later than those who did not. The Borrelia load in the ticks did not explain the risk of seroconversion. Regional and sex differences in the Borrelia seroprevalence were found. The risk of developing a Borrelia infection after a bite by a Borrelia-infected tick is small but increases with the duration of tick feeding.

Comparison of molecular responses based on BCR-ABL1%IS results from an in-house TaqMan-based qPCR versus Xpert® assay in CML patients on tyrosine kinase inhibitor therapy

Objectives:Tyrosine kinase inhibitors (TKIs) have drastically changed the prospects for chronic myeloid leukemia (CML) patients. The European LeukemiaNet (ELN) recommends molecular monitoring of BCR-ABL1 mRNA levels at distinct time points to define an optimal response, warning, or failure of treatment.Methods:Sixty-four follow-up peripheral blood samples from CML patients on TKI were tested by two methods. Molecular responses based on BCR-ABL1%IS from an Xpert® BCR-ABL1 Monitor assay were compared with TaqMan-based qPCR.Results:Seven samples showed ‘molecularly undetectable leukaemia’ by both methods (11%). In-house qPCR showed 57 BCR-ABL1+ samples; 45/57 samples (79%) were concordant for ‘major molecular response’ (MMR, n = 32) and ‘no MMR’ (n = 13) by both assays, whereas nine were BCR-ABL1 negative by Xpert®. Identical molecular responses (i.e. ‘optimal’) were defined in 41 samples. Discordances seen in patients < 10 months on TKI (n = 2) had no impact on clinical management, whereas for patients >12 months on TKI, a different molecular response was defined (‘warning’ versus ‘optimal’). Thirteen samples had ‘no MMR’ by both methods. 10/13 showed identical intervals (>10%IS, 1–10%IS or 0·1–1%IS), corresponding to seven ‘failures’ and three ‘warnings’. Discordant intervals were seen in 3/13 samples (all defined as ‘failures’). Deep molecular responses (MR4·0 or MR5·0) with detectable BCR-ABL1 showed some fluctuations between both methods, nevertheless, all had ‘optimal’ responses. ‘Molecularly undetectable leukaemia’ was observed more frequently by Xpert® (n = 16) as by our in-house assay (n = 7).Discussion:Based on current ELN recommendations, Xpert® BCR-ABL1 assay defines identical molecular responses as TaqMan-based qPCR BCR-ABL1%IS data in 98% (63/64) of samples.

Detection of human parvovirus 4 viremia in the follow-up blood samples from seropositive individuals suggests the existence of persistent viral replication or reactivation of latent viral infection.

BackgroundThe transmission routes for human parvovirus 4 (PARV4) infections in areas with high seroprevalence are not known. In the work described here, persistent PARV4 viral replication was investigated by conducting a longitudinal study.MethodsTen healthcare workers each provided a blood sample at the beginning of the study (first sample) and 12 months later (second sample). The paired samples were tested for PARV4-positivity by immunoblotting analysis and nested polymerase chain reactions.ResultsIgG antibodies against PARV4 were detected in six participants, three of whom also had IgM antibodies against PARV4. The immunoblotting results did not vary over time. PARV4 DNA was detected in the first blood sample from one participant who had IgG antibodies against PARV4 and in the second blood samples from 2 participants who had IgG and IgM antibodies against PARV4.ConclusionsDetection of PARV4 DNA in the second blood samples from two seropositive participants suggests the existence of persistent PARV4 replication or reactivation of inactive virus in the tissues. The finding of persistent or intermittent PARV4 replication in individuals with past infections provides an important clue toward unraveling the non-parenteral transmission routes of PARV4 infection in areas where the virus is endemic.

Long-term dynamics of gastric biomarkers after eradication of Helicobacter pylori infection.

Secretion of pepsinogen I (PgI), pepsinogen II (PgII), fasting gastrin-17 (fG-17) and stimulated gastrin-17 (sG-17) changes after Helicobacter pylori eradication. Few data are available on the long-term dynamics of gastric biomarkers after H. pylori eradication.The aim of this study was to investigate the dynamics of gastric biomarkers in H. pylori-positive patients after eradication over a 3-year period and to compare the levels with initially H. pylori-negative patients. Blood samples for the detection of gastric biomarkers were obtained from dyspeptic patients coming for upper gastrointestinal endoscopy. In H. pylori-positive patients, after eradication therapy, three follow-up blood samples were drawn after 12, 24 and 36 months; in H. pylori-negative patients, two samples were taken - at 12 and after 30 months. Median values of biomarkers in follow-up samples were compared with the baseline sample. The final sample included 110 patients (median age 67 years, M/F ratio 27/83). In patients after H. pylori eradication (n=83) PgI, PgII, fG-17 and sG-17 had decreased significantly during a 36-month period, whereas the PgI/PgII ratio had increased significantly from 5.59 to 11.64. In H. pylori-positive dyspeptic patients, after eradication therapy, a decrease in PgI, PgII, fG-17 and sG-17 was observed after 36 months whereas an increase in the PgI/II ratio suggested an improvement in gastric atrophy. The median levels of gastric biomarkers in patients after H. pylori eradication therapy may become similar to biomarker levels among initially H. pylori-negative individuals.

Repeated measurements of NT-pro-B-type natriuretic peptide, troponin T or C-reactive protein do not predict future allograft rejection in heart transplant recipients.

Studies on the prognostic value of serial biomarker assays for future occurrence of allograft rejection (AR) are scarce. We examined whether repeated measurements of NT-pro-B-type natriuretic peptide (NT-proBNP), troponin T (TropT) and C-reactive protein (CRP) predict AR. From 2005 to 2010, 77 consecutive heart transplantation (HTx) recipients were included. The NT-proBNP, TropT, and CRP were measured at 16 ± 4 (mean ± standard deviation) consecutive routine endomyocardial biopsy surveillance visits during the first year of follow-up. Allograft rejection was defined as International Society for Heart and Lung Transplantation (ISHLT) grade 2R or higher at endomyocardial biopsy. Joint modeling was used to assess the association between repeated biomarker measurements and occurrence of future AR. Joint modeling accounts for dependence among repeated observations in individual patients. The mean age of the patients at HTx was 49 ± 9.2 years, and 68% were men. During the first year of follow-up, 1,136 biopsies and concurrent blood samples were obtained, and 56 patients (73%) experienced at least one episode of AR. All biomarkers were elevated directly after HTx and achieved steady-state after ∼ 12 weeks, both in patients with or without AR. No associations were present between the repeated measurements of NT-proBNP, TropT, or CRP and AR both early (weeks 0-12) and late (weeks 13-52) in the course after HTx (hazard ratios for weeks 13-52: 0.96 (95% confidence interval, 0.55-1.68), 0.67 (0.27-1.69), and 1.44 (0.90-2.30), respectively, per ln[unit]). Combining the three biomarkers in one model also rendered null results. The temporal evolution of NT-proBNP, TropT, and CRP before AR did not predict occurrence of acute AR both in the early and late course of the first year after HTx.

Sequence Level Analysis of Hodgkin Lymphoma Clonotypes Detected in Peripheral Blood Using a Next-Generation Sequencing Approach

Background: Classical Hodgkin lymphoma (CHL) has been established as B-cell malignancy characterized by a clonal expansion of pathognomonic Reed Sternberg cells (Marafioti et al. Blood 2000). A previous report suggests that clonotypic B-cells may be present in the blood of patients with CHL; however, the relationship between these circulating clonotypic B-cells and CHL is unclear. We utilized the LymphoSIGHT™ method, a next-generation sequencing approach, to detect lymphoma-specific clonotypes in peripheral blood in patients with CHL at diagnosis or disease recurrence as well as in follow-up blood samples. This method has the sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood, and has been applied to minimal residual disease (MRD) detection in multiple B-cell malignancies. We evaluated the extent of somatic hypermutation in the lymphoma clonotypes, and performed sequence and expression level analyses of the lymphoma clonotype repertoire.Methods: Frozen primary tumor biopsy samples were first analyzed for clonality at the immunoglobulin heavy chain (IGH) and kappa chain (IGK) loci using the LymphoSIGHT method. Rearranged immunoglobulin gene segments (IGH-VDJ, IGH-DJ and IGK) in the genomic DNA and/or RNA were amplified with locus-specific primer sets, sequenced, and analyzed using standardized algorithms for clonotype determination. Clonotypes with a frequency >5% in the B-cell repertoire of the tumor biopsy were considered to represent tumor clonotypes. IGH-VDJ clonotypes with a frequency >2% in DNA were deemed to be cancer-specific if the clonotype was not present in the RNA. Such clonotypes were then quantitated in serum and peripheral blood mononuclear cells (PBMC), and DNA sequence and/or RNA expression level analysis was performed.Results: A total of 17 CHL patients were analyzed. A high-frequency clonal rearrangement was identified for at least one receptor (IGH-VDJ, IGH-DJ and IGK) in 12 of 17 cases (71%). Lymphoma-specific clonotypes were detected in blood samples from 8 of 11 patients (73%). Notably, a lymphoma-specific clonotype was detected in the serum compartment in 8 of 9 cases (89%) tested (Figure 1A), while it was detected in PBMC only in 3 of 9 cases (33%) tested (Figure 1B). Follow-up samples obtained from three patients in remission were negative for the tumor-specific clonotype in both the serum and cellular compartments. We conducted sequence and expression level analysis of each IGH-VDJ clonal rearrangement. We calculated the number of somatic mutations in each lymphoma-specific clonotype compared to the germline sequence in the interrogated region. In the ten patients with IGH-VDJ clonal rearrangements, we observed a median of 14 somatic mutations (range 0 to 27). This confirms that HRS cells correspond in their developmental stage to germinal or post-germinal center B-cells. While IGH-VDJ clonotypes were observed frequently in DNA obtained at diagnosis, IGH-VDJ clonotypes were not detected in the RNA from the same sample. We evaluated the relationship between the presence of lymphoma-specific clonotypes in the cellular compartment at diagnosis and eventual progression. All three untreated patients that were positive at baseline in the cellular compartment experienced relapse or progression (at 3, 11 and 17 months). In contrast, zero of 5 patients without detectable lymphoma-specific clonotypes in their cellular compartment at baseline experienced relapse (follow up duration 23-45 months, log-rank test p=0.004).Conclusions: This is the first clinical assay that can be used to detect and monitor MRD in CHL. Lymphoma-specific sequences can be identified in serum in 80% of cases. Our preliminary analysis suggests that the presence of lymphoma-specific clonotypes in PBMCs may indicate high risk for recurrence. This study demonstrates proof-of-principle and underscores the promise of a new methodology to measure disease burden and provide prognostic information from a blood test in patients with CHL. [Display omitted] [Display omitted] DisclosuresKlinger:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Kong:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Potential risk of blood transfusion‐transmitted brucellosis in an endemic area of China

Brucellosis is a severe zoonotic disease that is increasingly prevalent in north China. A study evaluating Brucella infection in blood donors was conducted at Kashi central blood station, Xinjiang, China. Four serologic and two molecular methods of detection of Brucella infection were used in plasma samples from blood donations collected from Kashi in northwest China, considered a brucellosis-endemic area. Blood donor samples collected in Shenzhen, southern China, a brucellosis-nonendemic area, were tested as a negative control group. In 3896 plasma samples collected from Kashi central blood station, 135 (3.5%) plasma samples were reactive by the Rose Bengal plate test (RBPT), and 120 (3.1%) of the 135 RBPT-reactive sample were also reactive with the standard tube agglutination test (SAT), respectively. All samples of the control group of 399 blood samples from Shenzhen blood center tested negative with RBPT and SAT. Of 135 seroreactive plasma samples, 39 (1.0%) reacted with B. melitensis membrane protein extracts by enzyme-linked immunosorbent assay and 25 were reactive to either rBP26 or rOMP31 by Western blot. Thirteen plasma samples and two follow-up blood samples were identified as carrying Brucella DNA by quantitative polymerase chain reaction (PCR) and nested PCR. Overall 15 (1:300) Kashi blood donations were found positive by nucleic acid testing, confirmed specific by DNA sequencing. The data indicate a probable high rate of Brucella bacteremia, suggesting a potential risk of transfusion-transmitted brucellosis. Blood donation screening for Brucella infection may be considered in the high Brucella-endemic areas of China.

Adipocyte cell size, free fatty acids and apolipoproteins are associated with non-alcoholic liver injury progression in severely obese patients

PurposeObesity is a modern pandemic with continuous expansion and represents an independent risk factor for non-alcoholic fatty liver disease (NAFLD), the most common liver disease in westernized countries. The crosstalk between adipose tissue and the liver is key to the development of NAFLD. ProceduresTherefore, in an observational study blood, visceral adipose tissue and liver tissue were obtained from 93 severely obese patients with a mean age of 43years and mean BMI of 52kg/m2 at the time of weight loss surgery. In a subset of patients a follow-up blood sample was obtained 6weeks after surgery to assess acute effects of weight loss. In addition to routine parameters of liver injury, serum samples were analyzed for leptin, adiponectin, free fatty acids (FFAs), and several apolipoproteins. Main findingsThe diameter of visceral adipocytes correlated to liver injury, serum markers of inflammation and serum adipokine levels. Liver injury assessed by serology (ALT, AST) and histology (NAFLD activity score, NAS) was independent of the BMI. However, serum levels of triglycerides and Apolipoprotein CIII (ApoCIII) were associated with NAS. Serum levels and composition of FFAs, especially long chain FFAs, also correlated with NAS. Analysis of serum samples six weeks after surgery revealed beneficial changes in serum triglycerides, levels of ApoCIII and several FFAs. ConclusionsIn severely obese patients beneficial effects on liver injury can been observed as early as six weeks after bariatric surgery. These effects may be explained by the observed changes in adipose tissue and lipid metabolism. Collectively, these findings underline the importance of the link between adipose tissue and the liver.

High rate of A(H1N1)pdm09 infections among rural Thai villagers, 2009-2010.

BackgroundPandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated.MethodsA cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008–2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection.ResultsThe 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts (70.6%, 12/17) occurred among children born between 1990 and 2003.ConclusionSubclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains.

Adiponectin receptor and adiponectin signaling in human tissue among patients with end-stage renal disease.

Adiponectin plasma levels in chronic kidney disease (CKD) are two to three times higher than in individuals with normal kidney function. Despite adiponectin's anti-diabetic, anti-inflammatory and anti-atherogenic properties, patients with CKD have insulin resistance, systemic inflammation and accelerated atherogenesis. Hence, although adiponectin production is increased by adipose tissue in end-stage renal disease (ESRD), it is unclear if its effects on metabolism remain intact. To determine if there is adiponectin resistance in ESRD, we measured tissue levels of adiponectin receptor-1 (AdipoR1) and adiponectin downstream effectors in ESRD patients compared with normal kidney function controls. Blood and tissue samples were obtained from participants at the time of kidney transplantation or kidney donation. A follow-up blood sample was obtained 3-6 months after transplantation. AdipoR1 was higher in muscle and peripheral blood mononuclear cells collected from ESRD patients. There was also a nonsignificant increase in AdipoR1 in visceral fat of ESRD compared with controls. Compared with controls, phosphorylation of the adiponectin downstream effector adenosine monophosphate-activated protein kinase (AMPK) was higher in ESRD while acetyl-CoA carboxylase phosphorylation (ACC-P) and carnitine palmitoyl transferase-1 (CPT-1) levels were lower. In vitro, exposure of C2C12 cells to uremic serum resulted in upregulation of AdipoR1 and increased phosphorylation of AMPK but decreased ACC-P and CPT-1 expression. Both our in vivo and in vitro observations indicate that uremia results in upregulation of AdipoR1 but adiponectin resistance at the post-receptor level.

JC Virus in CD34+and CD19+Cells in Patients With Multiple Sclerosis Treated With Natalizumab

Infection with JC virus (JCV) may lead to development of demyelinating progressive multifocal leukoencephalopathy in patients with multiple sclerosis (MS) who are treated with natalizumab. To determine whether mononuclear cells in circulation from MS patients treated with natalizumab harbor JCV DNA. In this prospective investigation, we enrolled 49 MS patients from the Clinical Center for Multiple Sclerosis at The University of Texas Southwestern Medical Center and 18 healthy volunteers. We drew 120-mL blood samples from 26 MS patients at baseline and at approximately 3-month intervals to 10 months during the course of natalizumab infusions. One blood sample was drawn from 23 MS patients receiving natalizumab for more than 24 months and from 18 healthy volunteers. Natalizumab treatment of MS. The blood samples were separated using flow cytometry into CD34+, CD19+, and CD3+ cell subsets; DNA templates were prepared using quantitative polymerase chain reaction for JCV DNA identification. Plasma samples were tested for anti-JCV antibodies by enzyme-linked immunosorbent assays performed at the Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological and Communicative Diseases and Stroke. Thirteen of the 26 patients (50%) with baseline and follow-up blood samples had detectable viral DNA in at least 1 cell compartment at 1 or more points. Ten of the 23 patients (44%) receiving treatment for more than 24 months and 3 of the 18 healthy volunteers (17%) also had detectable viral DNA in 1 or more cell compartment. Fifteen of the 49 MS patients (31%) were confirmed to harbor JCV in CD34+ cells and 12 of 49 (24%) in CD19+ cells. Only 1 of 18 healthy volunteers were viremic in CD34+ cells and none in CD19+ cells. Nine patients and 1 healthy volunteer were viremic but had seronegative test results for JCV antibodies. JC virus DNA was detectable within cell compartments of natalizumab-treated MS patients after treatment inception and longer. JC virus DNA may harbor in CD34+ cells in bone marrow that mobilize into the peripheral circulation at high concentrations. Latently infected cells initiate differentiation to CD19+ cells that favors growth of JCV. These data link the mechanism of natalizumab treatment with progressive multifocal leukoencephalopathy.

BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn.

BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn.

Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: A prospective analysis

Chronic lymphocytic leukemia (CLL) patients may acquire new chromosome abnormalities during the course of their disease. Clonal evolution (CE) has been detected by conventional chromosome banding (CBA), several groups also confirmed CE with fluorescence in situ hybridization (FISH). At present, there are minimal prospective data on CE frequency determined using a combination of both methods. Therefore, the aim of our study was to prospectively assess CE frequency using a combination of FISH and CBA after stimulation with CpG oligonucleotides and interleukin-2. Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE using FISH and CBA after stimulation. Patients provided baseline and regular follow-up peripheral blood samples for testing. There was a median of 3 cytogenetic examinations (using both methods) per patient. CE was detected in 15.7% (22/140) of patients using FISH, in 28.6% (40/140) using CBA, and in 34.3% (48/140) of patients by combining both methods. Poor-prognosis CE (new deletion 17p, new deletion 11q or new complex karyotype) was detected in 15% (21/140) of patients and was significantly associated with previous CLL treatment (p=0.013). CBA provides more complex information about cytogenetic abnormalities in CLL patients than FISH and confirms that many patients can acquire new abnormalities during the course of their disease in a relatively short time period.