EDTA Blood Research Articles

Cell-free fetal DNA as a non-invasive method using pyrosequencing in detecting beta-globin gene mutation: A pilot study from area with limited facilities in Indonesia.

BackgroundThalassemia is a monogenic, autosomal recessive, inherited disorder of the red blood cells caused by mutations or deletions in the globin gene. Approximately 6–10% of the Indonesian population carries the β-globin gene mutation; however, premarital screening is rarely conducted, and antenatal screening is optional. We explored the use of cell-free fetal DNA (cffDNA) as a potential non-invasive method of detecting the fetal β-globin gene mutation prenatally in pregnant women.Materials and methodsPregnant mothers (n = 10), who were known carriers of thalassemia and who had a history of having borne a baby with thalassemia major, and their carrier husbands (n = 4) were recruited after providing consent. EDTA blood was drawn, and maternal DNA, including cffDNA, and paternal DNA were isolated. Maternal contamination tests were conducted using the variable number tandem repeat test for ApoB and D1S80 loci. Allele quantification was performed by pyrosequencing. Known mutations from the bio-archived DNA of patients with thalassemia major (n = 16) were run alongside as a control.ResultsIn total, 7 out of 10 cffDNA successfully passed the maternal contamination test. The results of the allele quantification showed that six fetuses were predictive carriers of IVS1nt5 and one was predictive normal, in line with the allele quantification for the bio-archived DNA from patients with thalassemia major. The minimum threshold percentage for mutant A allele at cd26 was 32%, mutant T allele at IVS1nt1 was 23%, and mutant C allele at IVS1nt5 was 39%.ConclusionTaking cffDNA from the mother’s blood proved useful as a non-invasive means of detecting the β-globin gene mutation using pyrosequencing allele quantification. This non-invasive method is of great interest for prenatal diagnosis in settings with limited facilities, as it minimizes the risk of abortion. Further study of other mutations of the β-globin gene is needed.

Phenotyping and Genotyping of HNA: Prevalence, Risk of Alloimmunization, and HNA Incompatibilities in Indians

Background: Antibodies to human neutrophil alloantigens (HNA) are involved in the pathophysiology of several clinical conditions including transfusion-related acute lung injury (TRALI), alloimmune and autoimmune neutropenia, and febrile nonhemolytic transfusion reactions leading to neutropenia. The cognate antigens are polymorphic structures expressed on several glycoproteins on the neutrophils, i.e., antigens HNA-1a, -1b, -1c, and -1d on Fc-γ-receptor IIIb; HNA-2 on CD177; HNA-3a and -3b on choline transporter-like protein 2; HNA-4a and -4b on CD11b/αM subunit of the αMβ2-integrin (CD11b/CD18, Mac-1, CR3); and HNA-5a and -5b on αL-subunit (CD11a) of the αLβ2 integrin (CD11a/CD18), leukocyte function associated molecule (LFA)-1. Currently, there is a lacuna of diagnostic methods for detection of HNA in India. This study aimed to determine the HNA frequencies in Indians, estimate the risk of alloimmunization, and prepare typed neutrophil panels, which can be used to detect HNA antibodies in neutropenia cases. Material and Methods: EDTA blood samples were collected from random 1,054 blood donors. HNA-2 was phenotyped on fresh EDTA samples using FITC labelled monoclonal anti-CD177 by flowcytometry. HNA-1 (FCGR3B) genotyping was carried out by DNA sequencing and PCR-RFLP. Antigens of HNA-3 (SLC44A2) and HNA-5 (ITGAL) were genotyped by PCR-RFLP using TaqαI and Bsp1286I restriction enzymes, respectively, while HNA-4 (ITGAM) was genotyped by PCR-SSP. Results: Allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were found to be 0.433, 0.444, and 0.087, respectively. FCGR3B*01+*02+*03− was the most common genotype (33.78%). Ten individuals showed deficiency of FCGR3B individuals, while 23 showed hyperexpression, i.e., FCGR3B*01+*02+*03+. FCGR3B*04and *05 occurred with a frequency of 0.002 and 0.024. HNA-2 was found to be a high frequency antigen occurring in 98.8% population. Four percent individuals showed atypical expression of CD177 on their neutrophils. Allele frequencies of SLC44A2*01 and SLC44A2*02were 0.812 and 0.188, respectively, and that of ITGAM*01, ITGAM*02, ITGAL*01, and ITGAL*02 were 0.9546, 0.0454, 0.2372, and 0.7628, respectively. Conclusion: This is the first study in India to report the frequencies of HNA among blood donors. Typed neutrophil panels identified in the present study will enable us to investigate suspected cases of immune neutropenia in future.

Study of NFKB Polymorphism with HPV Infection in Women of Reproductive Age Group

Background: Human Papilloma Virus (HPV) is highly prevalent, with around 80% of women thought to be infected at some point. They are thought to produce proteins which inhibit the tumour suppressor protein p53 in cervical epithelial cells, allowing for uncontrolled cell division. The persistent HPV infection changes a premalignant lesion to cancerous state and this seems to be accompanied by a progressive loss of responsiveness to the NFƙB mediated growth inhibitory signal. This study was undertaken to provide a more precise evaluation of association of NFƙB -94ins/del ATTG promoter polymorphism and cancer risk. Methods: The study included 75 cases (unhealthy cervical pathology) and 75 controls (healthy normal cervix) belonging to reproductive age (15-44years) group. Cervical swab for HPV genotyping was performed in cobas x 480 and cobas z 480 analysers. DNA was extracted from EDTA blood sample using cobas DNA sample preparation kit. NFƙB gene polymorphism was seen by PCR-RFLP method in gel electrophoresis. Statistical analysis was done using SPSS 20. Results: Among the healthy normal cervical cytology, HPV infection rate was found to be 6.6%. In cases, maximum HPV positivity was for HPV 16. Among the study population, insertion allele was seen more in both the study groups compared to deletion allele. We found del/del genotype contribution maximum among the cases with cervical pathology. Ins/del genotype was found in cases group. In unhealthy cervix, we found that ins/ins genotype was prevalent among all the HPV strain except HPV 18 followed by del/del genotype. But, among HPV 18 strain, del/del was seen more common.

Development of a chromatographic lateral flow immunoassay for detection of African swine fever virus antigen in blood

African swine fever (ASF) is a highly lethal disease of domestic and wild swine caused by African swine fever virus (ASFV). The disease currently circulates in Africa, Europe, Asia and on the island of Hispaniola. The ongoing epizootics in Europe and Asia have produced millions of animal deaths and severe economic losses. No effective vaccine is available for ASF, making rapid and accurate detection of ASFV essential for disease mitigation strategies. Currently available diagnostics for ASFV possess significant limitations related to assay performance, deployability, and/or turn-around time; therefore there is an unmet need for pen-side diagnostic tests with sufficient sensitivity and specificity. A chromatographic lateral flow immunoassay (LFIA) was developed for the detection of ASFV antigen in EDTA-treated whole blood using monoclonal antibodies targeting the viral p30 protein. The assay requires only water to perform and provides results in 25 min, making it well-suited for field use. The LFIA was capable of detecting genotype I and genotype II strains of ASFV in EDTA blood from experimentally infected pigs at varying time-points after infection, though it was unable to detect a genotype X ASFV strain. Diagnostic sensitivity correlated with clinical disease severity, body temperature, and viral DNA levels, and was over 90% in animals showing moderate to severe ASF-related symptoms after challenge with virulent genotype II virus. The LFIA also showed a robust diagnostic specificity of over 98%, which is essential to field testing for a high consequence to foregin animal disease. The LFIA targeting the viral p30 protein can reliably detect ASFV in whole blood from animals showing moderate to severe clinical signs of infection with virulent genotype I and II isolates, making it a promising candidate for use as a field-deployable antigen detection assay. Additional evaluation using field samples and different virus strains is required to further assess the utility of this rapid diagnostic test.

Rickettsia massiliae circulation in sheep and attached Rhipicephalus sanguineus in Central Portugal.

Rickettsiosis is considered an emerging/re-emerging vector-borne disease that causes significant public health threats. Ticks are reservoirs and vectors of Rickettsia having a significant role in the transmission of rickettsiae. In Portugal, little is known about tick-borne Rickettsia species in sheep. Therefore, this study aimed to investigate rickettsiae infection in ticks and their sheep host from 27 farms in four districts of central Portugal, to clarify the role of the sheep host in the circulation of this zoonotic agent. Between March and May 2021, EDTA blood samples (n = 100) of healthy grazing sheep and their ticks (n = 100, one tick per animal) were collected during a herd health program in central Portugal. Obtained ticks were identified as Rhipicephalus sanguineus sensu lato by PCR targeting a partial sequence of 16S rRNA gene followed by sequence analysis. Rhipicephalus sanguineus s.l. and host sheep blood were tested for the presence of Rickettsia spp. by PCR targeting a partial sequence of ompB and ompA genes. From a total of 100 paired R. sanguineus s.l. and host sheep, Rickettsia massiliae was detected in 62 ticks and 35 grazing sheep blood samples, collected in central Portugal, 2021. All 35 positive sheep had attached positive R. sanguineus s.l., with matching nucleotidic sequences. These findings suggest that sheep may develop rickettsiemia and are likely capable of transmitting and amplifying the infection to uninfected ticks maintaining rickettsiae in circulation in the domestic cycle.

Immune response during regional chemotherapy.

e14537 Background: Regional chemotherapy is known to yield high drug concentrations in the treated tumor area while keeping systemic drug concentrations low. Regional Chemotherapy can be performed as an intra arterial infusion or isolated perfusion. Known perfusion techniques are isolated limb perfusion, hypoxic pelvic perfusion, hypoxic abdominal perfusion, upper abdominal perfusion, and isolated thoracic perfusion, Only little is known on the effect of chemotherapy on immune cells, especially not if chemotherapy is applied regionally. Since more and more immuno oncologic treatment possibilities arise, that often are combined with chemotherapy, knowledge on the effects on immune cells is crucial. Since systemic chemotherapy often decreases the number of immune cells in general, the more targeted approach of regional chemotherapy is investigated in this study. Methods: In this work, we investigate subpopulations of T-lymphocytes, Natural Killer cells and B-lymphocytes during regional chemotherapy and report on their changes after cycles of intra-arterial chemotherapy infusion followed by chemofiltration. Surface phenotyping of cells from EDTA blood was performed with combinations of monoclonal antibodies (anti- CD3,CD16, CD56, CD45, CD4, CD19, CD8, CD28, HLA-DR, CD39, CD25, CD127, CD45RA, CD45RO) for FACS analysis. 58 Patients with advanced breast cancer, ovarian cancer, head and neck cancer, lung cancer, or bladder cancer received regional chemotherapy as an intra arterial infusion therapy or an isolated perfusion therapy. Results: Contrary to the mild immune cell depletion during the first two cycles a more activated immune status is seen after treatment cycle three. Non-significant increase or stable numbers are observed for T-Lymphocytes and its subsets of CD4+ T-helper cells, CD8+ cytotoxic T-cells, and terminal memory T-cells (CD8+/CD28-/CD57+). Significant increase of cell numbers is observed for B-lymphocytes, regulative CD8+ T-cells (CD8+/CD28-/CD57-), activated HLA-DR+ T cells, and Natural Killer cells while regulative CD4+ T cells are significantly decreased. Conclusions: The data show that regional chemotherapy has controversial effects on the immune cell subpopulations. As every chemotherapy, it does affect immune cells, however to a limited extend, but importantly has an activating potential. Immune activation prevalently takes place after the third cycle of therapy which might be a hint for auto-vaccination caused by phases of repeated tumor necrosis after locally high drug concentrations. Finding the optimal dosage of chemotherapeutics may be crucial to balance direct tumor response, immune toxicity and immune activation.

Bluetongue Virus Infection of Goats: Re-Emerged European Serotype 8 vs. Two Atypical Serotypes.

In recent years, numerous atypical Bluetongue virus (BTV) strains have been discovered all around the world. Atypical BTV strains are phylogenetically distinct from the classical BTV serotypes 1–24 and differ in terms of several biological features. For the first time, the atypical strains BTV-25-GER2018 and BTV-33-MNG3/2016 as well as the re-emerged classical strain BTV-8-GER2018 were evaluated comparatively in a pathogenesis study in goats—the natural host of atypical BTV. A substantial number of in-contact animals were included in this study to detect potential contact transmissions of the virus. After infection, EDTA blood, ocular, nasal and oral swab samples as well as serum were collected regularly and were used for virological and serological analyses, respectively. Our study showed differences in the immunological reaction between the two atypical BTV strains (no group-specific antibody detection) and the classical BTV strain BTV-8-GER2018 (group-specific antibody detection). Furthermore, we observed an increase in the total WBC count (neutrophils and lymphocytes) in goats infected with the atypical BTV strains. No horizontal transmission was seen for all three strains. Our study suggests that the atypical BTVs used in the trial differ from classical BTVs in their immunopathogenesis. However, no evidence of direct contact transmission was found.

New Aspects of the Virus Life Cycle and Clinical Utility of Next Generation Sequencing based HIV-1 Resistance Testing in the Genomic, the Proviral, and the Viral Reservoir of Peripheral Blood Mononuclear Cells.

Typically, genotypic resistance testing is recommended at the start of antiretroviral therapy and is even mandatory in cases of virologic failure. The material of choice is plasma viral RNA. However, in patients with low viremia (viral load < 500 copies/ml), resistance testing by population-based sequencing is very difficult. Therefore, we aimed to investigate whether next generation sequencing (NGS) from proviral DNA and RNA could be an alternative. EDTA blood samples (n = 36) from routine clinical viral load testing were used for the study. Viral loads ranged from 96 to 390,000 copies/mL, with 100% of samples having low viremia. Distribution of subtypes; A (n = 2), B (n = 16), C (n = 4), D (n = 2), G (1), CRF02 AG (n = 5), CRF01 AE (n = 5), undefined/mixed (n = 4). The extracted consensus sequences were uploaded to the Stanford HIV Drug Resistance Data Base and Geno2pheno for online analysis of drug resistance mutations and resistance factors. A total of 2476 variants or drug resistance mutations (DRMs) were detected with Sanger sequencing, compared with 2892 variants with NGS. An average of 822/1008 variants were identified in plasma viral RNA by Sanger or NGS sequencing, 834/956 in cellular viral RNA, and 820/928 in cellular viral DNA. Both methods are well suited for the detection of HIV substitutions or drug resistance mutations. Our results suggest that cellular RNA or cellular viral DNA is an informative alternative to plasma viral RNA for variant detection in patients with low viremia, as shown by the high correlation of variants in the different viral pools. We show that by using UDS, a plus of two DRMs per patient becomes visible, which can make a big difference in the assessment of the expected resistance behavior of the virus.

A Study of Nuclear Factor-Kappa B1 Gene Polymorphism Types in Schizophrenia Patients and Their Correlation With Disease Severity.

Background and objectiveSchizophrenia is a chronic mental illness that is associated with multifactorial causation, but the greatest risk factor is a positive family history. Previous studies have suggested that proinflammatory cytokines and acute-phase proteins increase and modulate the severity of symptoms of schizophrenia. The inflammatory milieu in these patients has been found to be controlled by the transcription factor nuclear factor-kappa B1 (NF-κB1) in the inflammatory cells. In this study, we aimed to examine the correlation between polymorphism of the NF-κB1 gene and the severity of disease symptoms in schizophrenia patients.Materials and methodsThis was a case-control study conducted on 90 diagnosed cases of schizophrenia patients with 90 matched healthy volunteers as controls. DNA was extracted from EDTA blood samples and PCR was run, and the study of the NF-κB1 gene polymorphism rs28362691 (-94 ATTG ins/del) was performed by using restriction fragment length polymorphism (RFLP).ResultsWe observed the ins/ins genotype (78%) to be more prevalent among the study population. The del/del and ins/del genotypes were seen in 6.7% and 14.4% of schizophrenic patients respectively. The insertion allele was seen more than the deletion allele. Pearson’s correlation analysis showed a significant positive correlation between NF-κB1 levels and disease severity with an r-value of 0.471 and a p-value of 0.027.ConclusionWe found that in schizophrenia patients, the insertion allele was higher than the deletion allele and the ins/ins genotype was higher in frequency than the del/del and ins/del genotypes. There was a strong positive association between the insertion genotype and the severity of disease symptoms in schizophrenia patients.

Molecular detection and characterization of lumpy skin disease viruses from outbreaks in Thailand in 2021.

Lumpy skin disease (LSD) is one of the most important transboundary and emerging diseases in cattle. The disease causes significant economic losses in animal production and trade worldwide. The first LSD outbreak was recorded in March 2021, at Roi-Et province in the northeastern region of Thailand. Thereafter, the disease had rapidly spread into neighbouring provinces and throughout the country. The aim of the present study was to provide information regarding to the molecular detection and characterization of LSD viruses from outbreaks in Thailand in 2021. There were 1,748,112 susceptible and 604,404 affected animals (n = 588,512 [36.30%], beef cattle; n = 12,367 [15.74%], dairy cattle and n = 3524 [7.35%], buffaloes). The morbidity and mortality rates were 34.57% and 3.47%, respectively, and the case fatality rate was 10.05% (60,713 deaths). Based on real-time polymerase chain reaction results, the p32 gene of LSD virus (LSDV) was detected more frequently in skin nodule samples (54/77, 70.13%) than in nasal swabs (26/55, 42.57%) and EDTA blood (16/77, 20.78%) samples. Moreover, the copy number of the p32 gene was higher in skin nodule samples than in nasal swab and EDTA blood samples (cycle threshold value = 21.94 ± 0.62 vs. 31.52 ± 0.66 and 34.27 ± 0.32, respectively). Furthermore, 29 (53.70%) of 54 capripoxvirus-positive skin nodule samples were successfully isolated from Madin-Darby bovine kidney cells, and the cytopathic effect was observed 72 h after inoculation. Based on the phylogenetic trees of the GPCR, ANK and RPO30 gene sequences, the LSDV isolates from Thailand were distinct from both the LSDV-field and LSDV-vaccine groups and were closely correlated with the LSDV strains isolated from mainland China, Hong Kong territory and Vietnam in 2020. Additionally, they could be a potential virulent vaccine-recombinant LSDV strain.

PHENOTYPIC AND GENOTYPIC CHARACTERISTICS OF PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS TYPE 3 IN PEDIATRIC POPULATION IN PAKISTAN

OBJECTIVE: To determine the phenotypic and genotypic characteristics of progressive familial intrahepatic cholestasis (PFIC) type 3 in Pakistani children in a hospital setting. METHODS: This cross-sectional observational study was conducted at department of Pediatrics Gastroenterology &amp; Hepatology, The Children’s Hospital Lahore, Pakistan from October 2020 to March 2021. Patients of either sex under 16 years of age presenting with jaundice, pruritus, neonatal cholestasis or with chronic liver and gamma glutamyl transferase &gt;100 IU/ml were included in the study after taking informed consent by parents. For Molecular genetics 2ml blood in EDTA was sent to an international laboratory free of cost on research basis. Reports were assessed and levels were noted and genetic coding was also recorded. Data was entered and analyzed in SPSS version 22. Molecular data was interpreted with the help of clinical geneticist. RESULTS: Out of 34 children, 14 (41.2%) were males and 20 (58.8%) were females. Mean age of children was 6.71±3.10 years. Consanguinity was noted in 32 (94.1%) parents having positive family history in 24 (70.6%) cases. The most common mutation was c. 1783C&gt;T p.(Arg595*), noted in 12 (35.3%) cases, followed by c. 2861G&gt;T p.(Gly954 ASP) [8 (23.5%) cases], c. 153G&gt;A p.(Trp51) [3 (8.8%) cases], c. 1714 C&gt;T p.(Gln572*) c. 1906C&gt;T p. (Gln636), c. 3220G&gt;A p.(Gly1074Arg, c. 3433del p. (val1145Leufsx7) in 2 (5.9%) cases each, c. 3859 C&gt;T p.(1287Argext*) c. 88-91del p.(Lys30gly fsx7) and c. 1429c&gt;T p. (Gln477) in one (2.9%) case each. CONCLUSION: Children with PFIC type 3 have variable phenotypic and genotypic presentation.

Serum Iron Status of Pregnant Women in Central Hospital Sapele, South-South Nigeria

Background: Low iron status and Iron deficiency anemia are major prenatal complications predisposing pregnant women to preventable causes of premature delivery, intrauterine growth retardation and neonatal and prenatal death in Nigeria. Therefore, this study aims to evaluate and compare serum iron status in pregnant and non-pregnant women in Sapele South-South Nigeria. Materials and methods: A total of three hundred and sixty (360) subjects were enrolled for this study comprising of 78 non-pregnant women and 282 pregnant women. 5ml of venous blood were collected from participant into EDTA and plan containers respectively. The serum obtain from clotted sample was used for serum iron estimation using iron reduction method while EDTA blood was used for PCV and hemoglobin estimation.

NF-?? Pathway is a key modulator of BRCA-1 mutation induced chemo-Resistance in triple negative breast cancer

Triple-negative breast cancer (TNBC) is considered to be more aggressive and have a poorer prognosis than other types of breast cancer. The specific gene expression pattern between TNBC and other non-triple negative breast cancer (nTNBC) is yet to be resolved. Hence the present study was aimed to study the BRCA-1 status and measure the important tumor markers mRNA expression in peripheral blood samples of TNBC and other nTNBC subjects and compared with control subjects. EDTA blood samples were obtained from 25 CA (cancer) breast patients and 25 controls for DNA and RNA isolation. PCR-RFLP analysis was performed to check the BRCA-1 mutation in all the samples. Results showed that BRCA-1 mutation was present in all TNBC patients. Subsequently, quantitative RT-PCR was performed to detect the relative mRNA expression of NF-κβ, VEGFA, Caspase-3, and ERBB2 gene transcripts. Results showed that the transcript levels of NF-κβ, VEGFA, and ERBB2 were significantly up regulated in TNBC (p=0.0001) when compared to nTNBC and control. Transcript levels of caspase-3 were significantly down regulated (p=0.0001) in TNBC compared to nTNBC and control subjects. In conclusion, BRCA-1 mutation in TNBC patients confers resistance to chemotherapy activated by NF-κβ pathway and activation of adhesion and angiogenesis was associated with aggressive and poor prognosis in TNBC patients. Keywords: TNBC, Breast cancer, Cytokines, Tumour and gene expression.

Increased ShTAL1 IgE responses post-Praziquantel treatment may be associated with a reduced risk to re-infection in a Ghanaian S. haematobium-endemic community.

BackgroundEvidence from recent studies in Schistosoma mansoni-endemic areas show an age-associated immunity that is positively correlated with IgE titres to Schistosoma mansoni-specific tegumental allergen-like protein 1 (SmTAL1). The structural homology between SmTAL1 and the S. haematobium-specific TAL1 (ShTAL1) has been verified, yet it remains unclear whether similar age- and immune-associated trends characterize ShTAL1. This community-based intervention study was conducted to assess whether ShTAL1IgE responses post-treatment with praziquantel (PZQ) might be associated with a reduced risk to re-infection with S. haematobium.Methodology/Principal findingsThis study was conducted at Agona Abodom, Central Region, Ghana, and involved 114 participants aged 6 to 55 years. EDTA blood samples were collected at baseline and 7 weeks after PZQ treatment (Follow-up). Baseline and Follow-up titres of specific IgG1, IgG4, and IgE antibodies to the S. haematobium-specific adult worm antigen (ShAWA), the Sh-specific soluble egg antigen (ShSEA), and the Sh-specific tegumental-allergen-like 1 protein (ShTAL1) in plasma samples were measured using sandwich ELISA. Participants at both time points also provided stool and urine for helminth egg detection by microscopy. Prevalence of S. haematobium at baseline was 22.80%, and decreased to 3.50% at Follow-up. The egg reduction rate (ERR) was 99.87%. Overall plasma levels of ShTAL1-IgE increased 7 weeks post-PZQ treatment, and with increasing age; whiles S. haematobium infection prevalence and intensity decreased. For S. haematobium-infected participants who were egg-negative at Follow-up (N = 23), minimal median levels of ShTAL1-IgE were observed for all age groups prior to treatment, whilst median levels increased considerably among participants aged 12 years and older at Follow-up; and remained minimal among participants aged 11 years or less. In the univariate analysis, being aged 12 years or older implied an increased likelihood for ShTAL1-IgE positivity [12–14 years (cOR = 9.64, 95% CI = 2.09–44.51; p = 0.004); 15+ years (cOR = 14.26, 95% CI = 3.10–65.51; p = 0.001)], and this remained significant after adjusting for confounders [12–14 years (aOR = 22.34, 95% CI = 2.77–180.14; p = 0.004); ≥15 years (aOR = 51.82, 95% CI = 6.44–417.17; p < 0.001)]. Conversely, median ShTAL1-IgG4 titres were hardly detectible at Follow-up.Conclusions/SignificanceThese findings demonstrate that increased IgE levels to ShTAL1 7 weeks after PZQ treatment could be associated with a reduced risk to re-infection, and adds to the large body of evidence suggesting a protective role of the treatment-induced ShTAL1 antigen in schistosomiasis infections. It was also quite clear from this work that apart from being persistently S. haematobium-positive, elevated ShTAL1-IgG4 levels at Follow-up could be indicative of susceptibility to re-infection. These outcomes have important implications in vaccine development, and in shifting the paradigm in mass chemotherapy programmes from a ‘one-size-fits-all’ approach to more sub-group-/participant-specific strategies in endemic areas.

A Comparison of the Accuracy of Handheld Hemoglobinometer and Hematocrit Measurements for Detecting Plasma Leakage in Dengue Hemorrhagic Fever.

PurposePractical methods for detecting plasma leakage should be readily available in all areas where dengue is endemic. We compared the accuracy of measurements obtained with a handheld HemoCue® Hb 201 instrument used for hemoglobin point-of-care testing (Hb-POCT) with that of measurements of hematocrit (Ht) levels for detecting plasma leakage in dengue patients.Patients and MethodsWe performed both measurements using the HemoCue® Hb201 system and microhematocrit method on EDTA blood taken from dengue patients at three time points during their hospitalization. Ascites, pleural effusion, or gallbladder thickening determined through ultrasound examinations were considered the gold standard for determining dengue hemorrhagic fever (DHF) versus dengue fever (DF).ResultsClose agreement between Hb-POCT and Ht measurements was indicated by an r square value of 0.845 in a linear regression. The sensitivity results for distinguishing between DHF and DF at admission were similar for Hb-POCT (63.6%) and Ht (66.7%) (Kappa = 0.75) using the optimal cutoff point determined via ROC analysis. Delta differences (in percentage) for Hb-POCT and Ht between the highest and lowest values showed lower sensitivity (45.5% and 48.5%, respectively; Kappa 0.60) when the optimal cutoff point was applied. Recommended cutoffs of ≥20% to confirm plasma leakage provided a slightly higher sensitivity using Hb-POCT (18.2%) compared with the sensitivity obtained using Ht (15.2%) with Kappa value of 97.9%.ConclusionOur results showed that the accuracy of Hb POCT measurements was similar and not inferior to Ht measurements for detecting plasma leakage in patients with DHF. We recommend that further evaluations are conducted to determine the optimal cutoff point given the low sensitivity associated with using ≥20% Hb-POCT or Ht increases to determine hemoconcentration.

Improving the Quality of EDTA-treated Blood Specimens from Mice.

Nonterminal blood sampling in laboratory mice is a very common procedure. With the goal of improving animal welfare, different sampling sites and methods have been compared but have not achieved a consensus. Moreover, most of these studies overlooked the quality of blood specimens collected. The main preanalytical concern with EDTA-treated blood specimens for hematology analyses is platelet aggregation, which is known to cause analytical errors. Our objective was to find a nonterminal blood sampling method with minimal adverse effects on mice and few or no platelet aggregates. We tested and compared 2 collection sites, 4 sampling methods, and 3 antithrombotic drugs in 80 C57BL6/j male and female mice by evaluating platelet aggregates on blood smears and platelet, WBC, and RBC counts. In addition, the blood collection process was carefully evaluated, and adverse effects were recorded. Platelet aggregation was lower in specimens collected from the jugular vein than from the facial vein, with no effect of the sampling device or the presence of an antithrombotic additive. Highly aggregated specimens were significantly associated with lower platelet counts, whereas aggregation had no effect on WBC or RBC counts. Adverse events during sampling were significantly associated with more numerous platelet aggregates. The jugular vein is thus a satisfactory sampling site in mice in terms of both animal welfare and low platelet aggregation. Using antithrombotic agents appears to be unnecessary, whereas improving sampling conditions remains a key requirement to ensure the quality of EDTA-treated blood specimens from mice.

The Impact of Anticoagulation Agent on the Composition and Phenotype of Blood Leukocytes in Dromedary Camels.

For the analysis of several cellular biomarkers, blood samples are anticoagulated using different agents with different modes of action. However, for the most commonly used anticoagulants, EDTA and heparin, varying effects on blood components have been reported in different species. As little is known about the impact of anticoagulants on the immunological evaluation of camel leukocytes, the present study analyzed the leukogram, the immunophenotype, and the cell vitality of camel leukocytes separated from blood samples anticoagulated with EDTA or lithium heparin. Using flow cytometry and staining with monoclonal antibodies to several cell surface markers, the composition and immunophenotype of camel leukocytes separated from blood anticoagulated with EDTA or heparin were analyzed. In comparison to EDTA-anticoagulated blood, using lithium heparin as an anticoagulant resulted in reduced numbers of total leukocytes and reduced numbers of neutrophils, which led to a reduced neutrophil to lymphocyte ratio. The analysis of cell necrosis and apoptosis after the staining of leukocytes with the DNA-sensitive dye propidium iodide and the mitochondrial membrane potential probe JC1 revealed a higher fraction of necrotic neutrophils and higher fractions of apoptotic neutrophils and monocytes in heparin blood than in EDTA blood. In addition, monocytes from heparin blood showed higher expression levels of the cell surface markers CD14, CD163, and MHCII when compared to cells from EDTA blood. Similarly, in heparin blood, CD44 and CD172a were expressed higher on neutrophils, while CD11a was expressed higher on lymphocytes in comparison to cells from EDTA blood. The results of the current study indicate the importance of considering the type of anticoagulant when investigating the composition, vitality, and immunophenotype of camel leukocytes.

Optimized HPLC method to elucidate the complex purinergic signaling dynamics that regulate ATP, ADP, AMP, and adenosine levels in human blood.

ATP released into the bloodstream regulates immune responses and other physiological functions. Excessive accumulation of extracellular ATP interferes with these functions, andelevated plasma ATP levels could indicate infections and other pathological disorders. However, there is considerable disagreement about what constitutes normal plasma ATP levels. Therefore, we optimized a method to accurately assess ATP concentrations in blood. We found that rapid chilling of heparinized blood samples is essential to preserve in vivo ATP levels and that differential centrifugation minimizes inadvertent ATP release due to cell damage and mechanical stress. Plasma samples were stabilized with perchloric acid, etheno-derivatized, and delipidated for sensitive analysis of ATP and related compounds using high-performance liquid chromatography (HPLC) and fluorescence detection. We measured 33 ± 20nM ATP, 90 ± 45nM ADP, 100 ± 55nM AMP, and 81 ± 51nM adenosine in the blood of healthy human adults (n = 10). In critically ill patients, ATP levels were 6 times higher than in healthy subjects. The anticoagulant greatly affected results. ATP levels were nearly 8 times higher in EDTA plasma than in heparin plasma, while AMP levels were 3 times lower and adenosine was entirely absent in EDTA plasma. If EDTA blood was not immediately chilled, ATP, ADP, and AMP levels continued to rise, which indicates that EDTA interferes with the endogenous mechanisms that regulate plasma adenylate levels. Our optimized method eliminates artifacts that prevent accurate determination of plasma adenylates and will be useful for studying mechanisms that regulate adenylate levels and for monitoring of pathological processes in patients with infections and other diseases.

Quantitation of plasma angiotensin II in healthy Chinese subjects by a validated liquid chromatography tandem mass spectrometry method.

Quantitation of plasma angiotensin (Ang) II, the active mediator of the renin-angiotensin system, is challenging owing to its low physiological concentration. We report a validated liquid chromatography-mass spectrometry (LCMS) method to overcome this challenge. Ang II was extracted from EDTA plasma by an offline solid-phase extraction procedure with a Waters MAX μElution plate. LCMS quantitation was performed on the Waters TQS system, monitoring the 3+ ions of the peptide. The analytical performance of the LCMS method was validated. The stability of Ang II was studied with or without the presence of a protease inhibitor. Local reference intervals were established from 143 healthy normotensive subjects (57% female, 21-60 years old). The Ang II LCMS method had a measurable range of 3.3-700 pmol/L. The between-batch precision coefficient of variation was <7% over Ang II concentrations of 8.6-110 pmol/L. No significant matrix interference and carryover were observed. There was no significant difference in Ang II concentration in EDTA blood and plasma for at least 2h and 1h at room temperature, respectively. Ang II was stable for at least 1 year when stored at -80°C, with or without the protease inhibitor. Age-dependent Ang II reference intervals were established: 4.4-17.7pmol/L (21-30 years) and 3.9-12.8pmol/L (31-60 years). The present LCMS method is suitable for quantitation of plasma Ang II to study the renin-angiotensin system.

P058 IL-36 signaling in Crohn’s disease – characterization of a patient with an IL36RA mutation

Abstract Background The interleukin 36 (IL-36) family consists of three agonists (IL-36α, IL-36β, IL-36γ), which activate the NF-κB pathway and lead to the production of pro-inflammatory cytokines and the natural antagonist (IL-36RA), which inhibits IL-36 signaling. While it is known that IL-36 signaling is important in the skin, it has been only recently reported that IL-36 might play an important role in maintaining gut homeostasis and in the development of fibrosis (Scheibe et al., 2019). However, it is not clear how IL-36 signaling contributes to the development of intestinal inflammation and which cell types are involved in this process. To shed more light on the role of IL-36 in intestinal inflammation, we took the opportunity to perform an in-depth characterization of a Crohn’s disease patient with a mutation in IL36RA and identify cell subsets responding to IL-36 stimulation. Methods By whole exome-sequencing of EDTA blood we identified in a Crohn’s disease patient a heterozygous mutation in IL36RA (p.Ser113Leu), which we confirmed by Sanger sequencing. By ELISA, we quantified IL-36RA serum levels in comparison to other Crohn’s disease (CD) patients and healthy donors (HD). Finally, we stimulated peripheral blood mononuclear cells (PBMCs) of the IL-36RA patient, CD patients, and HDs with ionomycin/PMA, LPS or IL-36α and performed deep immune profiling by mass cytometry with 35 markers. Results Our IL36RA mutation carrying patient presented with a severe course of Crohn’s disease and consecutive of fibrosis, which was resistant to all commonly used treatment options. As an individual treatment, autologous stem cell transplantation was performed, but did not lead to long-term improvement in symptoms. By ELISA, we found strongly elevated IL-36RA levels in the serum of our patient compared to CDs and HDs. Mass cytometry analysis revealed that the IL36RA mutation does not lead to major shifts in the immune cell composition of the blood, as the frequencies of B, T, NK, and myeloid cells in PBMCs of our patient were comparable to those in HDs and CDs. Furthermore, our data revealed that IL-36α stimulation mainly induced TNFα and IL-6 expression in myeloid cells as well as IFNγ expression in NK cells but did not affect T and B cells. Conclusion Our data is the first description of a Crohn’s disease patient with an IL36RA mutation and indicate that defects in IL-36 signaling should be considered as a possible cause for therapy-refractory and fibrotic Crohn’s disease. Furthermore, we identified NK cells and myeloid cells as main responders to IL-36α stimulation. As next step, we plan to use recombinant produced, mutant IL-36RA in in-vitro assays with immune cells and fibroblasts to study the effect of the mutation on the IL-36RA function in more depth.