Expression of sPD-L1 levels in an ex vivo liver perfusion model.

The programmed cell death protein 1 (PD-1) acts as a central inhibitory immune checkpoint receptor. The soluble form of its primary ligand, sPD-L1, was found to be elevated in serum of patients with cancer, infectious diseases and chronic inflammation. So far, the hepatic origin of sPD-L1 has received relatively little attention and is therefore subject of this study in the context of normothermic machine perfusion (NMP) of liver grafts. sPD-L1 concentrations as well as several well-established clinically relevant laboratory parameters were determined in the perfusate of 16 donor liver grafts undergoing normothermic machine perfusion (NMP) up to 30 hours (h). sPD-L1 levels continuously increased during NMP and significantly correlated with markers of hepatic synthesis (cholinesterase), acute-phase proteins (von Willebrand factor, procalcitonin, antithrombin, interleukin-6, fibrinogen) and liver decay markers (gamma-glutamyltransferase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase). Perfusate leukocytes were in the lower reference range, and decreased after 12 h. Mean sPD-L1 levels in the perfusate correlated with donor levels of gamma-glutamyltransferase, alanine aminotransferase, creatinine and blood urea nitrogen. Our study reveals a significant increase of concentration of sPD-L1 following ischemia-reperfusion injury in a hepatic ex vivo model. sPD-L1 concentrations during NMP correlate with established acute-phase proteins and liver cell decay markers suggesting that hepatic sPD-L1 synthesis or shedding increases during the acute phase and cell decay. Furthermore, sPD-L1 correlates with established liver function and synthesis parameters as well as with donor laboratory values and might therefore be a potential biomarker for hepatic function of liver grafts.

Anti-Thrombin IgA in a Patient with Multiple Myeloma Leading to In Vitro Interference in Multiple Coagulation Tests and Confounding Diagnosis.

BACKGROUND: We report the case of a 59-year-old multiple myeloma patient in whom an anti-human thrombin IgA antibody led to prolonged in vitro coagulation times, suggesting inhibitors to all intrinsic coagulation factors in the absence of spontaneous bleeding. METHODS: Routine and extensive special coagulation tests, in vivo bleeding time, and specific antibody testing were performed. RESULTS: Although the patient did not suffer from spontaneous bleeding and had a normal in vivo bleeding time, the anti-human thrombin IgA autoantibody affected all coagulation assays involving human thrombin in vitro, mimicking inhibitors to intrinsic coagulation factors. As the IgA paraprotein and the IgA antibody virtually disappeared after autologous stem cell transplantation, the coagulation tests also largely normalized. CONCLUSION: Antibodies to human thrombin may interfere with all coagulation assays involving thrombin, imitating a severe coagulopathy. However, in vivo they do not necessarily lead to strongly increased bleeding tendency. Complex and ambiguous coagulation abnormalities should be evaluated and treated in an interdisciplinary setting, including a highly specialized coagulation laboratory, from the beginning.

Serum sPD-L1 levels are elevated in patients with viral diseases, bacterial sepsis or in patients with impaired renal function compared to healthy blood donors.

OBJECTIVES: Immune checkpoints play an important role in maintaining the balance of the immune system and in the development of autoimmune diseases. A central checkpoint molecule is the programmed cell death protein 1 (PD-1, CD279) which is typically located on the surface of T cells. Its primary ligand PD-L1 is expressed on antigen presenting cells and on cancer cells. Several variants of PD-L1 exist, among these soluble molecules (sPD-L1) present in serum at low concentrations. sPD-L1 was found elevated in cancer and several other diseases. sPD-L1 in infectious diseases has received relatively little attention so far and is therefore subject of this study. METHODS: sPD-L1 serum levels were determined in 170 patients with viral infections (influenza, varicella, measles, Dengue fever, SARS-CoV2) or bacterial sepsis by ELISA and compared to the levels obtained in 11 healthy controls. RESULTS: Patients with viral infections and bacterial sepsis generally show significantly higher sPD-L1 serum levels compared to healthy donors, except for varicella samples where results do not reach significance. sPD-L1 is increased in patients with impaired renal function compared to those with normal renal function, and sPD-L1 correlates significantly with serum creatinine. Among sepsis patients with normal renal function, sPD-L1 serum levels are significantly higher in Gram-negative sepsis compared to Gram-positive sepsis. In addition, in sepsis patients with impaired renal function, sPD-L1 correlates positively with ferritin and negatively with transferrin. CONCLUSIONS: sPD-L1 serum levels are significantly elevated in patients with sepsis, influenza, mesasles, Dengue fever or SARS-CoV2. Highest levels are detectable in patients with measles and Dengue fever. Also impaired renal function causes an increase in levels of sPD-L1. As a consequence, renal function has to be taken into account in the interpretation of sPD-L1 levels in patients.

Evaluation of two fully automated ADAMTS13 activity assays in comparison to manual FRET assay.

INTRODUCTION: The objective of the present study was to evaluate and compare the validity and utility of two fully automated ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity assays for clinical diagnostic decision-making and to compare their performance. METHODS: Two automated ADAMTS13 activity assays (Werfen HemosIL® AcuStar ADAMTS13 Activity, Technoclone Technofluor ADAMTS13 Activity) were compared with a manual FRET assay (BioMedica ACTIFLUOR ADAMTS13 Activity). The following samples were used: 13 acute phase TTP (thrombotic thrombocytopenic purpura) samples from 11 different patients, one sample from a patient with congenital ADAMTS13 deficiency, 16 samples from control patients, three follow-up samples from TTP patients in long-term remission and one sample from a patient with stem cell transplantation related thrombotic microangiopathy (TMA). The WHO 1st International Standard for ADAMTS13 and several dilutions of normal plasma with ADAMTS13-depleted normal plasma were also tested. Statistical analysis included descriptive statistics, sensitivity and specificity, Passing & Bablok regression and Bland-Altman plot. RESULTS: The quantitative comparison between the HemosIL® (x) and Technofluor (y) methods showed a strong correlation (Pearson r = 0.98, n = 49). When considering an ADAMTS13 activity of <10% as a hallmark for the diagnosis of TTP, two fully automated assays were both able to identify all TTP- and non-TTP-samples correctly, resulting in sensitivities and specificities of 100%. CONCLUSION: Both fully automated ADAMTS13 activity assays showed a good diagnostic performance and quantitative correlation among themselves, discriminating reliably between TTP- and non-TTP-patients.

One-Year Follow-Up of COVID-19 Patients Indicates Substantial Assay-Dependent Differences in the Kinetics of SARS-CoV-2 Antibodies.

Determination of antibody levels against the nucleocapsid (N) and spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are used to estimate the humoral immune response after SARS-CoV-2 infection or vaccination. Differences in the design and specification of antibody assays challenge the interpretation of test results, and comparative studies are often limited to single time points per patient. We determined the longitudinal kinetics of antibody levels of 145 unvaccinated coronavirus disease 2019 (COVID-19) patients at four visits over 1 year upon convalescence using 8 commercial SARS-CoV-2 antibody assays (from Abbott, DiaSorin, Roche, Siemens, and Technoclone), as well as a virus neutralization test (VNT). A linear regression model was used to investigate whether antibody results obtained in the first 6 months after disease onset could predict the VNT results at 12 months. Spike protein-specific antibody tests showed good correlation to the VNT at individual time points (rS, 0.74 to 0.92). While longitudinal assay comparison with the Roche Elecsys anti-SARS-CoV-2 S test showed almost constant antibody concentrations over 12 months, the VNT and all other tests indicated a decline in serum antibody levels (median decrease to 14% to 36% of baseline). The antibody level at 3 months was the best predictor of the VNT results at 12 months after disease onset. The current standardization to a WHO calibrator for normalization to binding antibody units (BAU) is not sufficient for the harmonization of SARS-CoV-2 antibody tests. Assay-specific differences in absolute values and trends over time need to be considered when interpreting the course of antibody levels in patients. IMPORTANCE Determination of antibodies against SARS-CoV-2 will play an important role in detecting a sufficient immune response. Although all the manufacturers expressed antibody levels in binding antibody units per milliliter, thus suggesting comparable results, we found discrepant behavior between the eight investigated assays when we followed the antibody levels in a cohort of 145 convalescent patients over 1 year. While one assay yielded constant antibody levels, the others showed decreasing antibody levels to a varying extent. Therefore, the comparability of the assays must be improved regarding the long-term kinetics of antibody levels. This is a prerequisite for establishing reliable antibody level cutoffs for sufficient individual protection against SARS-CoV-2.

Intermittent retinal artery occlusions as the first clinical manifestation of polycythemia vera: a case report.

BACKGROUND: Polycythemia vera (PV) is a myeloproliferative neoplasm with increased hemoglobin, hematocrit, platelet count and leukocytosis, resulting in increased blood viscosity. PV which is initially presenting with ocular symptoms is rare, but irreversible retinal vessel occlusions leading to the diagnosis of PV have been described in literature. CASE PRESENTATION: We describe a patient with PV, initially presenting with attacks of monocular temporary loss of vision due to intermittent retinal artery occlusions of different retinal arteries. The patient was immediately treated with phlebotomy and the impaired arterial retinal perfusion could be restored without permanent retinal ischemia. We were able to document these transient arterial occlusions with fundus photography as well as fluorescein angiography. To the best of our knowledge, a case like this has never been documented before. CONCLUSION: This report is pertinent, in order to raise awareness among clinicians for polycythemia vera, as it can in fact be used as a differential diagnosis for patients with retinal artery occlusion. We would like to stress that early therapy might reverse the vessel complications.

Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

Hepatitis D virus (HDV) prevalence in Austria is low but causes considerable morbidity due to fast progression to cirrhosis.

BACKGROUND: Hepatitis D virus (HDV) coinfection aggravates the course of hepatitis B virus (HBV). The prevalence of HDV in Austria is unknown. OBJECTIVE: This national study aimed at (i) recording the prevalence of HDV-infection in Austria and (ii) characterizing the "active" HDV cohort in Austria. METHODS: A total of 10 hepatitis treatment centers in Austria participated in this multicenter study and retrospectively collected their HDV patients between Q1/2010 and Q4/2020. Positive anti-HDV and/or HDV-RNA-polymerase chain reaction (PCR) results were retrieved from local database queries. Disease severity was assessed by individual chart review. Viremic HDV patients with clinical visits in/after Q1/2019 were considered as the "active" HDV cohort. RESULTS: A total of 347 anti-HDV positive patients were identified. In 202 (58.2%) patients, HDV-RNA-PCR test was performed, and 126/202 (62.4%) had confirmed viremia. Hepatocellular carcinoma was diagnosed in 7 (5.6%) patients, 7 (5.6%) patients underwent liver transplantation, and 11 (8.7%) patients died during follow-up. The "active" Austrian HDV cohort included 74 (58.7%) patients: Evidence for advanced chronic liver disease (ACLD, i.e., histological F3/F4 fibrosis, liver stiffness ≥10 kPa, varices, or hepatic venous pressure gradient ≥6 mmHg) was detected in 38 (51.4%) patients, including 2 (5.3%) with decompensation (ascites/hepatic encephalopathy). About 37 (50.0%) patients of the "active" HDV cohort had previously received interferon treatment. Treatment with the sodium-taurocholate cotransporting polypeptide inhibitor bulevirtide was initiated in 20 (27.0%) patients. CONCLUSION: The number of confirmed HDV viremic cases in Austria is low (<1% of HBV patients) but potentially underestimated. Testing all HBV patients will increase the diagnostic yield. More than half of viremic HDV patients had ACLD. Improved HDV testing and workup strategies will facilitate access to novel antiviral therapies.

Clinical validation of the Siemens quantitative SARS-CoV-2 spike IgG assay (sCOVG) reveals improved sensitivity and a good correlation with virus neutralization titers.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections cause coronavirus disease 2019 (COVID-19) and induce a specific antibody response. Serological assays detecting IgG against the receptor binding domain (RBD) of the spike (S) protein are useful to monitor the immune response after infection or vaccination. The objective of our study was to evaluate the clinical performance of the Siemens SARS-CoV-2 IgG (sCOVG) assay. METHODS: Sensitivity and specificity of the Siemens sCOVG test were evaluated on 178 patients with SARS-CoV-2-infection and 160 pre-pandemic samples in comparison with its predecessor test COV2G. Furthermore, correlation with virus neutralization titers was investigated on 134 samples of convalescent COVID-19 patients. RESULTS: Specificity of the sCOVG test was 99.4% and sensitivity was 90.5% (COV2G assay 78.7%; p<0.0001). S1-RBD antibody levels showed a good correlation with virus neutralization titers (r=0.843; p<0.0001) and an overall qualitative agreement of 98.5%. Finally, median S1-RBD IgG levels increase with age and were significantly higher in hospitalized COVID-19 patients (median levels general ward: 25.7 U/mL; intensive care: 59.5 U/mL) than in outpatients (3.8 U/mL; p<0.0001). CONCLUSIONS: Performance characteristics of the sCOVG assay have been improved compared to the predecessor test COV2G. Quantitative SARS-CoV-2 S1-RBD IgG levels could be used as a surrogate for virus neutralization capacity. Further harmonization of antibody quantification might assist to monitor the humoral immune response after COVID-19 disease or vaccination.

Evaluation of four commercial, fully automated SARS-CoV-2 antibody tests suggests a revision of the Siemens SARS-CoV-2 IgG assay.

OBJECTIVES: Serological tests detect antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the ongoing coronavirus disease-19 (COVID-19) pandemic. Independent external clinical validation of performance characteristics is of paramount importance. METHODS: Four fully automated assays, Roche Elecsys Anti-SARS-CoV-2, Abbott SARS-CoV-2 IgG, Siemens SARS-CoV-2 total (COV2T) and SARS-CoV-2 IgG (COV2G) were evaluated using 350 pre-pandemic samples and 700 samples from 245 COVID-19 patients (158 hospitalized, 87 outpatients). RESULTS: All tests showed very high diagnostic specificity. Sensitivities in samples collected at least 14 days after disease onset were slightly lower than manufacturers' claims for Roche (93.0%), Abbott (90.8%), and Siemens COV2T (90.3%), and distinctly lower for Siemens COV2G (78.8%). Concordantly negative results were enriched for immunocompromised patients. ROC curve analyses suggest a lowering of the cut-off index for the Siemens COV2G assay. Finally, the combination of two anti-SARS-CoV-2 antibody assays is feasible when considering borderline reactive results. CONCLUSIONS: Thorough on-site evaluation of commercially available serologic tests for detection of antibodies against SARS-CoV-2 remains imperative for laboratories. The potentially impaired sensitivity of the Siemens COV2G necessitates a switch to the company's newly filed SARS-CoV-2 IgG assay for follow-up studies. A combination of tests could be considered in clinical practice.

Comparative evaluation of four SARS-CoV-2 antigen tests in hospitalized patients.

OBJECTIVES: Rapid identification of infected subjects is a cornerstone for controlling a pandemic like the current one with the SARS-CoV-2. Easy to handle antigen tests can provide timely results, which is of particular importance in a primary care setting. However, concerns exist regarding their sensitivity, which led us to evaluate four commercially available tests in patients hospitalized for COVID-19. METHODS: We analyzed in parallel nasopharyngeal/oropharyngeal swabs from 154 consecutive patients admitted to our department with moderate to severe COVID-19, using quantitative RT-PCR (Cobas, Roche) and up to four antigen tests from different distributors. Antigen test results were linked to Ct (cycle threshold) values as markers for patients' infectivity. RESULTS: We found that two out of four antigen tests correctly identified subjects with high viral loads (Ct≤25), and three out of four tests detected more than 80% of subjects with a Ct≤30, which is considered the threshold for infectivity. However, one test investigated had a poor clinical performance. When investigating subjects with Ct values >30, we found that the antigen test was still positive in up to 45% of those cases. CONCLUSION: Most antigen tests had a sufficient sensitivity to identify symptomatic subjects infected with SARS-CoV-2 and with transmissible infection. On the other hand, antigen testing may not be suitable to identify loss of infectivity in COVID-19 subjects during follow-up. Newly introduced antigen tests need to be validated in a clinical or primary care setting to define their clinical usefulness.

Plasma phosphatidylcholines and vitamin B12/folate levels are possible prognostic biomarkers for progression of Alzheimer's disease.

OBJECTIVES: In clinical practice it is important to identify patients suffering from mild cognitive impairment (MCI) who will progress to Alzheimer's disease (AD). The purpose of this study is to investigate whether lipid metabolites and vitamin B12 and folate levels are effective biomarker for an accurate prediction of MCI-to-AD conversion. METHODS: During the standard diagnostic assessment at our memory clinic 48 cognitively healthy subjects and MCI patients were recruited. These participants were followed up after 7-9 years. Blood was collected, various biochemical markers (including vitamin B12 and folate) analysed and plasma lipids were measured using the AbsoluteIDQ p150 Kit. RESULTS: There was no significant change in lipid levels in controls converting to MCI. However, we found significant changes in five lipids in converters from controls to AD. Interestingly, also two lipids were altered when MCI re-converted to controls. Vitamin B12 levels were not affected by conversion but folate levels significantly decreased in MCI-AD conversion. CONCLUSIONS: Taken together, our study provides evidence that some plasma lipids are significantly altered in subjects converting to AD. Future studies will investigate whether the peripheral lipid changes correspond with changes in the brain during the course of the disease. Although this is a small study, there are indications that lipids may be suitable as prognostic markers.

Treatment of Concurrent Thrombotic Thrombocytopenic Purpura and Graves' Disease: A Report on Two Cases.

Graves' disease (GD) and thrombotic thrombocytopenic purpura (TTP) are autoimmune diseases caused by autoantibodies against the TSH receptor (TRAb) and the enzyme ADAMTS13. We here report on two patients with concurrent GD and TTP, who achieved sustained remission of both conditions with the TTP treatment regimen and thiamazole. Both patients suffered from relapsing TTP and were diagnosed with GD concomitantly at the time of relapse. They were treated with steroids, plasma exchange, rituximab, and thiamazole. This therapy induced complete remission of TTP. TRAb levels also decreased rapidly and both patients developed subclinical hypothyroidism three and five weeks later. Our observations suggest that TTP and GD may be concomitant and that GD possibly triggers a relapse of TTP. The combination of thyrostatic treatment and immunosuppression with PE, rituximab, and steroids is able to induce rapid and prolonged remission of GD.

Genotyping of coeliac-specific human leucocyte antigen in children with type 1 diabetes: does this screening method make sense?

OBJECTIVES: Due to a high linkage disequilibrium of diabetes and coeliac-specific human leucocyte antigen (HLA) genotypes, the prevalence of coeliac disease (CD) in children and adolescents with diabetes mellitus type 1 (T1D) is much higher than in the general population. Recently, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) revised new screening guidelines in which genotyping for coeliac-specific HLA alleles is recommended for high-risk patients as patients with T1D. The aim of our study was to investigate the frequency and distribution of coeliac-specific HLA genotypes in paediatric patients with T1D. STUDY DESIGN: HLA genotyping was performed on paediatric patients with T1D, recruited at the Medical University Hospital of Innsbruck and Graz. The test was done by PCR. Statistical analysis was performed with IBM-SPSS V.20. RESULTS: In 121 paediatric patients with T1D (52% male), mean age 13.3 (SD 3.9) years, mean age at diabetes diagnosis 7.4 (SD 3.8) and mean diabetes duration of 5.9 (SD 3.3) years, HLA genotyping was conducted. Ninety-two per cent showed positive HLA DQ2 and/or HLA DQ8 genotypes. Thirty-four per cent carried HLA DQ2, 33% were HLA DQ2+DQ8 positive and 25% of the patients showed positive results for HLA DQ8 alone. Only 8% had no coeliac-specific HLA markers. Four (3%) patients were diagnosed with CD. CONCLUSIONS: The majority of paediatric patients with T1D has positive coeliac-specific HLA genotypes DQ2 and/or DQ8. Therefore, genotyping for coeliac-specific HLA alleles as a first-line test in patients with T1D as recommended in the ESPGHAN guidelines does not seem reasonable. Screening for coeliac-specific antibodies needs to be performed on a regular basis for patients with T1D.

Predicting Transfusion Requirements During Extracorporeal Membrane Oxygenation.

OBJECTIVE: Patients requiring extracorporeal membrane oxygenation (ECMO) have a well-known bleeding risk and the potential for experiencing possibly fatal thromboembolic complications. Risk factors and predictors of transfusion requirements during ECMO support remain uncertain. The authors hypothesized that compromised organ function immediately before ECMO support will influence transfusion requirements. DESIGN: A prospective observational study. SETTING: A tertiary, single-institutional university hospital. PARTICIPANTS: The study included 40 adult patients requiring ECMO for intractable cardiac and respiratory failure between July 2010 and December 2012. Blood samples were taken before initiation of ECMO (baseline), after 24 and 48 hours on ECMO, and 24 hours after termination of ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Independent of veno-arterial or veno-venous support, 26% of patients required≥2 packed red blood cells per day (PRBC/d) and 74% of patients required<2 PRBC/d during ECMO. Requirements of≥2 PRBC/d during ECMO support were associated with higher creatinine levels and lower prothrombin times (PT, %) at baseline and with impaired platelet function after 24 hours on ECMO. Platelet function, activated by thrombin receptor-activating peptide stimulation, decreased by 30% to 40% over time on ECMO. Receiver operating characteristic curve analysis showed cut-off values for creatinine of 1.49 mg/dL (sensitivity 70%, specificity 70%; area under the curve [AUC] 0.76, 95% confidence interval [CI] 0.58-0.94), for PT of 48% (sensitivity 80%, specificity 59%; AUC 0.69, 95% CI 0.50-0.87), and for thrombin receptor-activating peptide (TRAP) 32 U (sensitivity 90%, specificity 68%; AUC 0.76, 95% CI 0.59-0.93). CONCLUSIONS: The results of this study demonstrated that increased creatinine levels and lower PT before ECMO and secondary impaired platelet function significantly increased transfusion requirement.