Comprehensive antibody and cytokine profiling in hospitalized COVID-19 patients in relation to clinical outcomes in a large Belgian cohort.

The immune response in patients with Coronavirus Disease 2019 (COVID-19) is highly variable and is linked to disease severity and mortality. However, antibody and cytokine responses in the early disease stage and their association with disease course and outcome are still not completely understood. In this large, multi-centre cohort study, blood samples of 434 Belgian COVID-19 hospitalized patients with different disease severities (ranging from asymptomatic/mild to critically ill) from the first wave of the COVID-19 pandemic were obtained. Baseline antibody and cytokine responses were characterized and associations with several clinical outcome parameters were determined. Anti-spike immunoglobulin (Ig)G and IgM levels were elevated in patients with a more severe disease course. This increased baseline antibody response however was associated with decreased odds for hospital mortality. Levels of the pro-inflammatory cytokines IL-6, IP-10 and IL-8, the anti-inflammatory cytokine IL-10 and the antiviral cytokines IFN-α, IFN-ß and IFN-λ1 were increased with disease severity. Remarkably, we found significantly lower levels of IFN-λ2,3 in critically ill patients compared to patients of the moderate and severe disease category. Finally, levels of IL-8, IL-6, IP-10, IL-10, IFN-α, IFN-ß, IFN-γ and IFN-λ1 at baseline were positively associated with mortality, whereas higher IFN-λ2,3 levels were negatively associated with mortality.

Vertical transmission of SARS-CoV-2 infection and preterm birth.

Viral infections are common complications of pregnancy, with a wide range of obstetric and neonatal sequelae. Currently, there are limited data on whether SARS-CoV-2 is vertically transmitted in pregnant women tested positive for the virus. Here we describe a case of a known SARS-CoV-2-positive woman giving preterm birth to two fetuses with SARS-CoV-2 positive testing in placental tissue and amniotic fluid. The placental histological examinations showed chronic intervillositis and extensive intervillous fibrin depositions with ischemic necrosis of the surrounding villi.

Hemoglobin S monitoring on TOSOH G8 in hemoglobin A1c mode in case of urgent red blood cell exchange.

BACKGROUND: Pre- and post-transfusion hemoglobin S (HbS) levels are used to document the efficacy of red blood cell exchange (RCE) in patients with sickle cell disease (SCD). In case of urgent RCE a 24/7 short turn-around time (STAT) analysis, with the ability to identify and quantify HbS, is warranted. The use of TOSOH G8 (Tosoh Europe) is evaluated for this purpose, using the variant HbA1c mode. METHODS: Analytical performance of the HbS analysis on TOSOH G8 in variant HbA1c mode was evaluated, including assessment of imprecision and linearity for HbS. In addition, a comparison study between TOSOH G8 and Minicap Flex Piercing (FP) system CZE (Sebia) using 32 HbS samples (HbS range: 9%-93%) was carried out to evaluate analytical and clinical concordance. RESULTS: Total HbS imprecision was 1.77% and 0.31% for a sickle cell trait and a sickle cell anemia sample, respectively. An acceptable linearity (HbS range: 6%-88%) was observed (R2  > .99). Passing-Bablok regression analysis showed a significant proportional bias; however, a good analytical concordance (r > .95) was found. Our results suggested that TOSOH G8 underestimated HbS results compared with those of Minicap FP system (mean difference: -3.54%), especially in samples with a high HbS concentration. CONCLUSION: Hemoglobin S results obtained with TOSOH G8 in variant HbA1c mode are clinically acceptable to monitor urgent RCE. The observed underestimation will not alter clinical decision-making.

Evaluation of the V8 E-Class, a Novel Automated Capillary Isoelectric Focusing Instrument for Hemoglobinopathy Screening.

OBJECTIVES: We evaluated the performance of a novel capillary isoelectric focusing (CIEF) application for hemoglobinopathy screening on the recently introduced V8 E-Class platform. METHODS: Analytical performance of the V8 E-Class was evaluated and included assessment of hemoglobin A2 (HbA2) imprecision; linearity for HbA2, fetal hemoglobin (HbF), and sickle hemoglobin (HbS); and carryover for HbS. Furthermore, a method comparison with the Minicap Flex Piercing (Sebia, Lisses, France), the Variant Classic (Bio-Rad Laboratories, Hercules, CA), and the G8 (Tosoh Europe, Amsterdam, the Netherlands) was done to assess analytical and clinical concordance. RESULTS: Total HbA2 imprecision was 3.26% and 3.14% for normal and elevated HbA2 controls and 5.16% and 3.58% for a normal and a heterozygous HbS patient sample, respectively. HbA2, HbF, and HbS showed acceptable linearity, and no carryover was observed. The method comparison showed good analytical concordance (r > 0.95) except for a homozygous HbS subset (r = 0.532-0.704). A comparable phenomenon was seen for the clinical concordance with good agreement in samples without variants (weighted κ > 0.80) but poorer agreement in HbS samples (κ < 0.30). CONCLUSIONS: Good analytical performance was demonstrated for this novel CIEF application for hemoglobinopathy screening. Method comparison showed generally good correlation but highlights the need for standardization. Finally, software optimization could further add to its use for routine hemoglobinopathy screening.

Hb Melusine and Hb Athens-Georgia: potentially underreported in the Belgian population? Four cases demonstrating the lack of detection using common CE-HPLC methods either for glycated hemoglobin (HbA1C) analysis or Hb variant screening.

OBJECTIVE AND IMPORTANCE: Suspected hemoglobin (Hb) variants, detected during HbA1C measurements should be further investigated, determining the extent of the interference with each method. CLINICAL PRESENTATION: This is the first report of Hb Melusine and Hb Athens-Georgia in Caucasian Belgian patients. Intervention & Technique: Since common CE-HPLC methods for HbA1C analysis or Hb variant screening are apparently unable to detect these Hb variants, their presence might be underestimated. HbA1C analysis using CZE, however, alerted for their presence. Moreover, in case of Hb Melusine, even Hb variant screening using CZE was unsuccessful in its detection. CONCLUSION: Fortunately, carriage of Hb Melusine or Hb Athens-Georgia variants has no clinical implications and, as shown in this report, no apparent difference in HbA1C should be expected.

Failing blood gas measurement due to methemoglobin forming hemoglobin variants: a case report and review of the literature.

INTRODUCTION: We present a case of an arterial blood gas sample analysis from a 33-year old woman where no oximetry results could be obtained using the Radiometer ABL800 FLEX device. Clinical history of this patient learned that she was carrier of a methemoglobin forming hemoglobin variant type Hyde Park (HbM Hyde Park) and raised the question whether or not this variant could be the cause of the errors obtained during analysis. MATERIALS AND METHODS: A literature search was performed, focusing on methemoglobin forming hemoglobin variants and their influence on oxygenation measurements. An overview of the currently described methemoglobin forming hemoglobin variants is also included. RESULTS AND DISCUSSION: In the presence of dyshemoglobins such as methemoglobin, techniques used to obtain parameters that reflect the patient oxygenation status, such as pulse oximetry and CO-oximetry can be influenced. In these cases, CO-oximetry is the preferred technique because it can compensate for this, in contrast to pulse oximetry. In case of the presence of methemoglobin originating from a hemoglobin variant, it is possible that CO-oximetry data cannot be calculated because the absorbance spectrum of this methemoglobin can differ from regular methemoglobin. Moreover, pulse oximetry devices are actually prone to erroneous results since pulse oximetry data will be calculated in these cases, but unreliable and should be avoided. CONCLUSION: Methemoglobin forming hemoglobin variants are rare genetic mutations. However, they can possibly interfere with the calculation of CO-oximetry values. In these cases, pulse oximetry data should be avoided because they could lead to incorrect medical decisions.

A Mosaic Expression of a Hb J-Amiens (HBB: c.54G > T; p.Lys18Asn) and its Interference with Hb A1c Analysis.

We report the case of a 56-year-old Caucasian woman in whom hemoglobinopathy screening was triggered following an aberrant Hb A1c analysis. Preliminary diagnosis of the hemoglobin (Hb) variant was obtained through cation exchange high performance liquid chromatography (HPLC) and gel electrophoresis. DNA analysis confirmed the presence of Hb J-Amiens [ß17(A14)Lys→Asn; HBB: c.[54G > C or 54G > T)]. However, an unbalanced ratio between wild type and mutant signal after direct sequencing and a lower than expected percentage of this Hb variant led to the suggestion of a mosaic expression. Furthermore, different methods [capillary zone electrophoresis (CZE), cation exchange HPLC and boronate affinity] were tested to study the possible interference of this variant with Hb A1c measurements. These investigations showed a clinically relevant difference between the methods tested. Hb A1c analysis may lead to the discovery of new Hb variants or mosaicism for previously described Hb variants. This may have genetic consequences for the offspring of carriers and brings about the question of partner testing.

Novel LC-MS/MS method for plasma vancomycin: comparison with immunoassays and clinical impact.

BACKGROUND: Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment. As literature suggests between-method differences, our first objective was to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for total vancomycin in human plasma and to compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of between-method quantification differences. METHODS: For LC-MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the internal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC-MS/MS as a reference. RESULTS: A novel LC-MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min was developed. The assay had a total imprecision of 2.6-8.5%, a limit of quantification of 0.3 mg/L and an accuracy ranging from 101.4 to 111.2%. Using LC-MS/MS as reference, three immunoassays showed a mean proportional difference within 10% and one showed a substantial mean proportional difference of >20%. Clinical discordant interpretation of the obtained concentrations ranged from 6.1 to 22.2%. CONCLUSIONS: We developed a novel LC-MS/MS method for rapid analysis of total vancomycin concentrations in human plasma. Correlation of the method with immunoassays showed a mean proportional difference >20% for one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples.

Evaluation of (99m)Tc-labeled tropanes with alkyl substituents on the 3beta-phenyl ring as potential dopamine transporter tracers.

Technetium(V)-oxo-3beta-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl[N-(2-mercaptoethyl), N-(N'-(2-mercaptoethyl)-2-aminoethyl)]-aminomethyl ((99m)Tc-TRODAT-1) and three derivatives with one or two substituents on the 3beta-phenyl ring (4-methylphenyl, 4-ethylphenyl and 2,4-dimethylphenyl) were prepared and evaluated as potential imaging agents for the central nervous dopamine transporter (DAT). Labeling of the ligands with (99m)Tc yielded for each of them a mixture of two radiolabeled species, which were purified and isolated using reversed-phase high-performance liquid chromatography. Employing radio-LC-MS, we found both species to have the same molecular mass suggesting diastereoisomers. After intravenous injection in mice and rats, the compounds were stable in vivo and no important metabolites were found in plasma or urine. Replacement of the 4-chloro atom on the 3beta-phenyl ring by a methyl group causes no loss of affinity for the DAT system. However, substitution of an ethyl group for the 4-chloro atom or introduction of a second methyl group in the 2-position of the phenyl ring results in a serious reduction of the affinity for the DAT transporter. Ex vivo autoradiography on mice brain slices and biodistribution studies in rats showed specific uptake of (99m)Tc-TRODAT-1 and the 4-methylphenyl derivative in striatum and putamen. Although the 4-ethylphenyl and 2,4-dimethylphenyl derivatives show brain uptake in rats and mice, no specific uptake in striatum was found. In addition, differences in biological behavior between the different diastereomers were observed. In conclusion, small changes to (99m)Tc-TRODAT-1 at the phenyl ring in the 3beta position of the tropane moiety significantly change the biological behavior of the studied compounds.

Biological evaluation of a technetium-99m-labeled integrated tropane-BAT and its piperidine congener as potential dopamine transporter imaging agents.

INTRODUCTION: Recently, we have reported modification of (99m)Tc-TRODAT-1 by integrating the N2S2 metal chelating unit and the tropane skeleton. Results of a preliminary biodistribution study in rats were promising with respect to brain uptake. The present report deals with the further biological characterization of the (99m)Tc-labelled integrated TRODAT derivatives ((99m)Tc-TropaBAT and (99m)Tc-norchloro-TropaBAT) and with the synthesis and biological evaluation of a novel (99m)Tc-labelled piperidine-based derivative ((99m)Tc-PipBAT). METHODS: Biodistribution of all radiolabelled complexes was studied in normal mice. A more detailed ex vivo intracerebral distribution study of the two (99m)Tc-TropaBAT complexes was additionally performed in normal rats. Autoradiography of brain sections of normal mice (with or without pretreatment with FP-beta-CIT or haloperidol) and rats was performed. Affinity for the dopamine transporter (DAT) was also assessed in vitro in the presence or absence of cocaine. RESULTS: Both (99m)Tc-TropaBAT complexes show a slightly higher brain uptake than (99m)Tc-TRODAT-1, but the striatum/cerebellum activity ratio is less favourable. Nevertheless, significant striatal uptake was detected after ex vivo autoradiography, but this uptake was also observed after pretreatment with FP-beta-CIT. Unexpectedly, no striatal uptake was detected after in vitro incubation of mouse brain sections with the tracer agents. For (99m)Tc-PipBAT, neither brain uptake nor in vitro striatal uptake was found. CONCLUSION: Both (99m)Tc-TropaBAT complexes exhibit similar diffusion into brain as (99m)Tc-TRODAT-1, and ex vivo autoradiography shows significant striatal uptake. However, the inferior striatum/cerebellum activity ratio, the striatal uptake in mice pretreated with FP-beta-CIT or haloperidol, and the lack of striatal uptake during in vitro incubation prove that the DAT is not targeted. Brain uptake disappears when the tropane skeleton is replaced by a piperidine ring, and also in this case no striatal uptake is found in vitro.

Synthesis and biological evaluation of a technetium-99m(I)-tricarbonyl-labelled phenyltropane derivative.

A new tropane derivative was synthesized by combining a tridentate ligand, N-(2-picolylamine)-N-acetic acid (2-PAA), and a phenyltropane derivative. It was labelled with a [(99m)Tc(CO)(3)](+) moiety, resulting in the formation of two stable and neutral lipophilic isomers. Their identity was confirmed using radio-LC-MS. In normal mice, no brain uptake was observed for any of the isomers and in vitro autoradiography using mouse brain sections showed no specific uptake in the striatal area.

Development and biological evaluation of 99mTc-BAT-tropane esters.

Two (99m)Tc-BAT-tropane conjugates, i.e., technetium(V)-oxo-3-[N-(2-mercaptoethyl), N-(N'-(2-mercaptoethyl)-2-aminoethyl)]-aminopropyl 3beta-(4-chlorophenyl)-8-methyl-8-azabicyclo [3.2.1]octane-2beta-carboxylate and the corresponding norchloro derivative, were prepared and evaluated as potential imaging agents for the central nervous dopamine transporter (DAT) system. In these compounds, a tropane and a (99m)Tc-BAT moiety were linked through an ester bond. Both compounds were formed as a mixture of two diastereomers which could be purified and isolated using reversed-phase high-performance liquid chromatography (HPLC). Radio-LC-MS analysis supported the hypothesised structure of the synthesised technetium complexes. After intravenous injection in mice and rats, the compounds were stable in vivo, and no important metabolites were found in plasma or urine. In vitro testing suggested specific competitive binding to the DAT system, but in vivo experiments in rats showed no significant brain uptake for the diastereomers of both compounds; neither was there any specific uptake in the striatum. The results suggest that replacement of a methylene linker by an ester does not seriously affect the binding properties of the tropane conjugates to the dopamine transporter but results in a drastic reduction of passage over the blood-brain barrier (BBB).

Technetium-99m labelled integrated tropane-BAT as a potential dopamine transporter tracer.

To reduce the molecular weight of 99mTc-labelled tropanes with the aim to enhance the passage over the blood-brain barrier, a so-called integrated tropane-BAT construct was developed. For this purpose a mercaptoethyl substituent was attached to the amine nitrogen atom of a nortropane precursor and the methyl carboxylate in 2beta-position was converted to a 2-mercaptoethylaminomethylene substituent. This integrated tropane-BAT construct could be labelled efficiently (85-90%) with technetium-99m. Results of LC-MS analysis of the tracer agent support the assumed structure. Biodistribution studies in normal rats (n=3) showed a slightly higher brain uptake for the new tracer agents as compared to 99mTc-TRODAT-1. These results indicate that further biological evaluation of the integrated 99mTc-tropane-BAT is warranted.

Optimization of the preparation of 99mTc-labeled Hynic-derivatized Annexin V for human use.

Hydrazino nicotinate (Hynic) is one of the most attractive bifunctional agents designed for the labeling of proteins with (99m)Tc. Recently, a (99m)Tc-labeled Hynic-Annexin V derivative has been described and successfully evaluated in animal models of apoptosis. Prior to a phase I human study, the preparation of (99m)Tc-Hynic-Annexin V has been optimized. The influence of the Hynic-load of Annexin V, amount of protein, nature and amount of reducing agent, activity and co-ligand on the labeling yield were evaluated using ITLC and size-exclusion FPLC. Optimal labeling yields were obtained when 60-90 microgram Hynic-Annexin V was labeled with up to 1.11 GBq (30 mCi) (99m)TcO(4)-using 10-20 microgram SnCl(2).2H(2)O as reducing agent and 1.5 mg tricine as the co-ligand. Biodistribution in normal mice was comparable to literature data.

Identity confirmation of 99mTc-MAG3, 99mTc-sestamibi and 99mTc-ECD using radio-LC-MS.

Due to the low concentrations in which 99mTc-radiopharmaceuticals are obtained (4-40 ng/ml), confirmation of the identity of these tracer agents in the European Pharmacopoeia is generally performed only indirectly by assessment of their retention times on RP-HPLC. We have investigated whether it is possible to obtain more direct proof of the identity of technetium-99m labelled radiopharmaceuticals using radio-LC-MS. As representative examples, negatively charged 99mTc-MAG3, positively charged 99mTc-Sestamibi and neutral 99mTc-ECD were used. The three technetium-99m radiopharmaceuticals were prepared in several conditions to obtain variable relative amounts of radiochemical impurities and variable concentrations of the complexes (pico- to nanomolar). The preparations were analyzed on a reversed phase C18 HPLC column using a radio-LC-MS system equipped with a time of flight mass spectrometer with electrospray ionization in positive (99mTc-Sestamibi, 99mTc-ECD) or negative (99mTc-MAG3, 99mTc-ECD) mode. For each of the studied complexes, the main peak in the radiometric channel coincided with the expected molecular ion mass of the corresponding technetium complex in the mass spectrometer channel. The relative error on the measured accurate mass was in the range of 10 ppm. The identity of several radiochemical impurities of the three technetium complexes was also confirmed or established. It is concluded that radio-LC-MS can be a sensitive aid in quality control of 'no carrier added' radiopharmaceuticals.

Safety, biodistribution, and dosimetry of 99mTc-HYNIC-annexin V, a novel human recombinant annexin V for human application.

UNLABELLED: 99mTc-hydrazinonicotinamido (HYNIC)-annexin V is a novel tracer for in vivo imaging of apoptosis. The present study on humans was performed to investigate the safety of (99m)Tc-HYNIC-annexin V and to quantify the biodistribution and radiation dose. METHODS: Six healthy, male volunteers participated in the study. A dual-head gamma camera was used to acquire conjugate anterior and posterior views. Imaging started with a transmission scan using a (57)Co-flood source to obtain a map of the local thickness of the volunteer. Approximately 250 MBq of (99m)Tc-HYNIC-annexin V were injected intravenously, directly followed by a 30-min dynamic study. Whole-body scans were obtained at about 30 min, 3 h, 6 h, and 24 h after injection. Organ uptake was determined after correction for background, scatter, and attenuation. The MIRDOSE3.1 program was used to calculate organ-absorbed doses and effective dose. Signs of adverse effects were investigated by monitoring renal and liver function, hematology, blood coagulation, and vital signs (blood pressure, pulse, respiration rate, temperature, and electrocardiogram). RESULTS: The kidneys accumulated 49.7 +/- 8.1 percentage injected dose (%ID) at 3 h after injection; the liver, 13.1 +/- 1.0 %ID; the red marrow, 9.2 +/- 1.8 %ID; and the spleen, 4.6 +/- 1.6 %ID. More than 90% of the blood activity was cleared with a half-life of 24 +/- 3 min. The biologic half-life of the activity registered over the total body was long (69 +/- 7 h). Excretion of the activity was almost exclusively through the urine (22.5 +/- 3.5 %ID at 24 h), and hardly any activity was seen in the bowel or feces. Absorbed doses were found to be 196 +/- 31 micro Gy/MBq for the kidneys, 41 +/- 12 micro Gy/MBq for the spleen, 16.9 +/- 1.3 micro Gy/MBq for the liver, and 8.4 +/- 0.9 micro Gy/MBq for the red marrow. The effective dose was 11.0 +/- 0.8 micro Sv/MBq, or 2.8 +/- 0.2 mSv for the average injected activity of 250 MBq. No adverse effects were observed. CONCLUSION: (99m)Tc-HYNIC-annexin V is a safe radiopharmaceutical, having a favorable biodistribution for imaging of apoptosis in the abdominal as well as thoracic area with an acceptable radiation dose.