KLINISCH LABORATORIUM

CENTRUM KLINISCH LABORATORIUM

ARTIKELS

ABSTRACT 1

Combined oropharyngeal/nasal swab is equivalent to nasopharyngeal sampling for SARS-CoV-2 diagnostic PCR

Desmet T, De Paepe P, Boelens J, Coorevits L, et al. BMC Microbiology, 2021, 21(1), 31

ABSTRACT Early 2020, a COVID-19 epidemic became a public health emergency of international concern. To address this pandemic broad testing with an easy, comfortable and reliable testing method is of utmost concern. Nasopharyngeal (NP) swab sampling is the reference method though hampered by international supply shortages. A new oropharyngeal/nasal (OP/N) sampling method was investigated using the more readily available throat swab.

35 patients were diagnosed with SARS-CoV-2 by means of either NP or OP/N sampling. The paired swabs were both positive in 31 patients. The one patient who tested negative on both NP and OP/N swab on admission, was ultimately diagnosed on bronchoalveolar lavage fluid. A strong correlation was found between the viral RNA loads of the paired swabs (r = 0.76; P < 0.05). The sensitivity of NP and OP/N analysis in hospitalized patients (n = 28) was 89.3% and 92.7% respectively.

This study demonstrates equivalence of NP and OP/N sampling for detection of SARS-CoV-2 by means of rRT-PCR. Sensitivity of both NP and OP/N sampling is very high in hospitalized patients.

ABSTRACT 2

Non-lethal intoxication by ingestion of 50 castor beans: serial measurement of ricinine in blood, plasma and urine

Lefever S, Geerts I, Vermeulen E, Croes K, et al. Journal of Analytical Toxicology, 2021, 45(5), e8-e12, doi:10.1093/jat/bkaa139

ABSTRACT A 30-year-old woman presented to the emergency department 2 days after ingestion of 50 castor beans. Her symptoms on admission were vomiting, diarrhea, abdominal cramps, agitation and anxiety. Initial laboratory tests showed a slightly elevated C-reactive protein and mild liver and kidney dysfunction. The patient was transferred to the medium care unit of our hospital where she was observed for possible organ failure. During the next days, the kidney function improved and liver function started to recover. Four days after admission, the patient was transferred to the psychiatric ward. Urine, serum, plasma and whole-blood samples were analyzed for ricinine using a quantitative LC–MS-MS method. Initial values on admission (serum and urine) were very high in comparison with previously reported cases. Based on these values, the patient was monitored closely in the following days.

The patient made a full recovery, and during the course of hospitalization, concentrations of ricinine in plasma/serum, blood and urine gradually declined. The presence of ricinine in a patient’s blood or plasma is a proof of castor bean and, hence, ricin exposure.

However, based on this case and previously reported cases in literature, we can conclude that no clear correlation can be established between ricinine blood, plasma or urine levels and the severity of the intoxication. Clinicians should be aware of the potential danger of a ricin intoxication, and patients should be monitored closely for several days due to the unpredictable outcome of the intoxication.

ABTSTRACT 3

A G316A polymorphism in the ornithine decarboxylase gene promoter modulates MYCN-driven childhood neuroblastoma

Gamble LD, Purgato S, Van Maerken T, et al. Cancers, 2021, 13(8), 1807

ABSTRACT Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and

preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

ABSTRACT 4

Predictors and Dynamics of the Humoral and Cellular Immune Response to SARS-CoV-2 mRNA Vaccines in Hemodialysis Patients: A Multicenter Observational Study

Van Praet J, Reynders M, De Bacquer D, De Bel A, et al. Journal of the American Society of Nephrology, 32 (12), 2021, 3208-3220

ABSTRACT Preliminary evidence suggests patients on hemodialysis have a blunted early serological response to SARS-CoV-2 vaccination. Optimizing the vaccination strategy in this population requires a thorough understanding of predictors and dynamics of humoral and cellular immune responses to different SARS-CoV-2 vaccines.

This prospective multicenter study of 543 patients on hemodialysis and 75 healthy volunteers evaluated the immune responses at 4 or 5 weeks and 8 or 9 weeks after administration of the BNT162b2 or mRNA-1273 vaccine, respectively. We assessed anti-SARS-CoV-2 spike antibodies and T cell responses by IFN-γ secretion of peripheral blood lymphocytes upon SARS-CoV-2 glycoprotein stimulation (QuantiFERON assay) and evaluated potential predictors of the responses. Compared with healthy volunteers, patients on hemodialysis had an incomplete, delayed humoral immune response and a blunted cellular immune response. Geometric mean antibody titers at both time points were significantly greater in patients vaccinated with mRNA-1273 versus BNT162b2, and a larger proportion of them achieved the threshold of 4160 AU/ml, corresponding with high neutralizing antibody titers in vitro (53.6% versus 31.8% at 8 or 9 weeks, P<0.0001). Patients vaccinated with mRNA-1273 versus BNT162b2 exhibited significantly greater median QuantiFERON responses at both time points, and a larger proportion achieved the threshold of 0.15 IU/ml (64.4% versus 46.9% at 8 or 9 weeks, P<0.0001).

Multivariate analysis identified COVID-19 experience, vaccine type, use of immunosuppressive drugs, serum albumin, lymphocyte count, hepatitis B vaccine nonresponder status, and dialysis vintage as independent predictors of the humoral and cellular responses. The mRNA-1273 vaccine's greater immunogenicity may be related to its higher mRNA dose. This suggests a high-dose vaccine might improve the impaired immune response to SARS-CoV-2 vaccination in patients on hemodialysis.

PRESENTATIES/ CONGRESSEN

ABSTRACT 1

Whole transcriptome profiling of liquid biopsies from tumor xenografted mouse models enables specific monitoring of tumor-derived RNA

Deleu J, Vermeirssen V, Morlion A, Van Maerken T, et al. april 2021, Gent, België januari 2021, Amsterdam, Nederland

ABSTRACT Liquid biopsies enable disease diagnosis and treatment response monitoring. As compared to cell-free DNA, extracellular RNA is relatively unexplored. We aim to assess to which extent tumor RNA ends up in different blood fractions using mice engrafted with human tumor cells. This may unveil which compartment is best suited to probe tumor-derived extracellular transcriptomes.

We examined the circulating transcriptome in mice xenografted with SK-N-BE(2C) human neuroblastoma cells or a patient-derived breast cancer, also including non-tumor bearing control mice. Blood was collected from 5 mice per group via cardiac puncture, followed by preparation of platelets, platelet-rich, platelet-poor and platelet-free plasma. Using 60 µl input volumes, an established total RNA sequencing workflow to profile extracellular mRNA with synthetic spike-in RNA for calibration purposes, and a dedicated data processing pipeline to unambiguously distinguish human (tumor) from murine (host) mRNA, we charted the extracellular transcriptomes.

Despite the high RNA concentration variability among individual mice, we were able to detect human RNA in

all plasma fractions in a ratio that was on average ten to hundred times lower than murine RNA. The murine RNA concentration in plasma increased with increasing platelet concentration. In contrast, human RNA concentrations were relatively constant and did not support the recently proposed concept of preferential loading of tumor RNA in platelets. We detected around 2000 human tumor-derived protein-coding genes. Functional exploration of the resulting transcriptomes revealed various enriched gene sets and pathways, indicative of biological signal. In conclusion, we demonstrate that mice xenograft models can be used to specifically study tumor-derived extracellular RNA in liquid biopsies.

ABSTRACT 2

Substantial performance differences among RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

Van Maerken T, Van Paemel R, Verniers K, Yigit N, et al. maart 2021, Online meeting (VIB, België)

ABSTRACT The use of blood-based extracellular RNA (exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated 10 blood collection tubes, 3 time points between blood draw and downstream processing, and 8 RNA purification methods using the supplierspecified minimum and maximum biofluid input volumes. The impact of these pre-analytics is assessed by deep transcriptome profiling of both small and messenger RNA from healthy donors' plasma or serum. Experiments are conducted in triplicate (for a total of 276 transcriptomes) using 189 synthetic spike-in RNAs as processing controls. When comparing blood tubes, serum mRNA is remarkably very similar to EDTA plasma mRNA, in contrast to serum-derived small RNAs that are markedly different in biotype composition compared to their plasma counterparts. Furthermore, so-called blood preservation tubes do not stabilize RNA very well, as is reflected by increasing RNA concentration and number of detected genes over time, and by compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity. Among others, we note a 50-fold difference in mRNA yield and a 5-fold difference in the number of detected mRNAs. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated processing SOPs, representing paramount groundwork for future exRNA-based precision medicine applications.

ABSTRACT 3

Quantification and kinetic profiling of anti-RBD antibodies in COVID-19 patient serum and whole blood using a fiber optic SPR biosensor

Qu J-H, Leirs K, Maes W, Callewaert N, et al. 2021, Palm Springs, United States

ABSTRACT During the ongoing COVID-19 pandemic, serological tests have been proven useful in many different aspects, including evaluation of individual/community seroprevalence by measuring antibody responses. While the majority of serological tests have been used only for antibody quantification, SPR technology demonstrates clear advantages because of its capacity to reveal real-time binding kinetics of patient antibodies.

Here, we present LF FO-SPR serological bioassays for both quantification and kinetic profiling of all antibody isotypes against SARS-CoV-2 RBD in COVID-19 patient serum and whole blood samples. We immobilized His6-tagged RBD on the FO probe using cobalt (III)-nitrilotriacetic acid (Co(III)-NTA) chemistry, for a stable bioreceptor patterning, essential to detect target in complex sample matrices. The LF and sandwich bioassays were developed with 10 and 500-fold sample dilution, respectively. For the latter, we used 2 types of detection antibodies (i.e. goat anti-human (GAH) IgG and GAH polyvalent antibody targeting IgG, IgM and IgA) conjugated with gold nanoparticles (AuNPs) for sensitivity enhancement.

We tested 22 COVID-19 patient serum samples by sandwich bioassay and benchmarked it with ELISA, using the same bioassay components for each. The same patient serum samples were tested by FO-SPR LF bioassay. A commercial normal human serum (NHS) obtained before COVID-19 served as the negative control (NC).

Finally, we applied the established FO-SPRLF and sandwich bioassays in testing the level of anti-RBD antibodies in COVID-19 patient whole blood samples. Here, a mixture of 5 negative blood samples served as the NC. The SPR shift obtained after 30 min and SPR slope obtained within 30-120 s were calculated and used as the detection signals. The cutoff-1 and cutoff-2 values were calculated by summing the average and 3 or 10 times standard deviation (SD) of the NC, respectively. One-way analysis of variance (ANOVA) and Bonferroni multiple comparison test were performed in Matlab to identify statistical differences between the mean values, with ‘ns’ indicating non-significant difference. For correlation, the SPR shift was normalized by dividing the value with the mean of all the values for all samples. Pearson correlation coefficient (PCC) and intraclass correlation coefficient (ICC) were calculated in Matlab. First, we tested the level of anti-RBD IgG antibodies in 22 COVID-19 patient serum samples by an FO-SPR sandwich bioassay and ELISA, with the former capable of distinguishing more samples from the NC based on both ANOVA and cutoff values. Next, the same serum samples were assessed by FO-SPR LF bioassay, which is a rapid alternative (30 min) to sandwich bioassay (67 min) with a similar performance .

Moreover, this allowed direct insight into the level of all antibody isotypes, including kinetic profiling (i.e. antibody binding speed) revealed by SPR slope, the latter having a different profile compared to SPR shift. The robustness of LF bioassay was also verified by its excellent correlation with the sandwich bioassay.

Finally, we tested whole blood samples from 14 COVID-19 convalescent patients by FO-SPR LF and sandwich bioassays. They were similar in distinguishing the positive samples from the NC, which was further improved by testing undiluted blood samples directly by LF bioassay. Rapid FO-SPR LF bioassays are capable of inspecting COVID-19 patient serum and whole blood samples for both quantification and kinetic profiling of antibodies, putting this technology at the forefront of other serological tests.

The feasibility of directly testing (undiluted) blood samples could also greatly simplify sample preparation and shed light towards developing FO-SPR technology into true POC biosensors. ABSTRACT 4

Combined flowcytometric phenotyping and interferon release assay of sars-cov-2 reactive T cells in whole blood

Calcoen B, Callebaut K, Vandenbulcke A, Callewaert N, et al. 2021, Brussels, Belgium

ABSTRACT Our aim was to include flowcytometric assessment of SARS-CoV-2 specific T cell activation to a commercially available cytokine release assay on whole blood samples.

The EUROIMMUN SARS-CoV-2 interferon gamma release assay (IGRA) was performed according to the manufacturer’s instructions on whole blood samples from donors vaccinated against SARS-CoV-2 (n=6). In addition, the remaining pellet after centrifugation was resuspended in physiological buffer. Reconstituted samples were stained with a panel of fluorescently labeled monoclonal antibodies including anti-CD3, anti-CD4, anti-CD8 and anti-CD69. Following lysis of red blood cells, flowcytometric sample acquisition was performed. Specific interferon gamma release (mean = 1368 ± 937.8 mIU/mL) was found in whole blood from vaccinated donors upon stimulation with SARS-CoV-2 antigens. Furthermore, using our protocol, it was possible to reliably differentiate both CD4+ and CD8+ T cells in the reconstituted whole blood samples following IGRA. In addition, expression of the early T cell activation marker CD69 was significantly upregulated in both CD4+ and CD8+ T cells from vaccinated donors upon stimulation with SARS-CoV-2 antigens.

This study showed that flowcytometric profiling of T cell activation can be successfully implemented as additional read-out to a commercially available SARS-CoV-2 specific interferon gamma release assay.

ABSTRACT 5

Real-world monitoring of bnt162b2 vaccine-induced SARS-CoV-2 specific functional antibody response and RBD specific B cells in healthcare workers

. Calcoen B, Callebaut K, Vandenbulcke A, Callewaert N, et al. 2021, Ghent, Belgium

ABSTRACT SARS-CoV-2 is the causative virus of an unseen and still ongoing pandemic. Today, multiple vaccines are available within the armamentarium to reduce overall SARS-CoV-2 related mortality and infection rate. Vaccine efficacy studies often rely solely on quantification of serologic responses. Besides conventional serology, in this study we also considered the frequency of circulating vaccine-induced B cells specific for the receptor binding domain (RBD) of SARS-CoV-2. In addition, we address the neutralization efficacy of the vaccine-induced antibodies. Our aim was to describe in detail the vaccine-induced B-cell response in healthcare workers three months after receiving COVID-19 vaccine bnt162b2. In a cohort of 30 healthcare workers from a supraregional hospital in Belgium, blood was taken before and three months after bnt162b2 vaccination against SARS-CoV-2. Serum was used to determine both anti-S IgG and anti-RBD IgG titers and to quantify the neutralization efficacy (in vitro inhibition of RBD binding to human ACE2). In addition, peripheral blood mononuclear cells were fluorescently labeled with anti-CD19 and RBD-biotin combined with streptavidin-PE to allow detection of circulating RBD specific B-cells. Local EC approval was obtained.

Three months post-vaccination, all subjects showed detectable vaccine-induced anti-S IgG (mean index: 26.06 ± 7.002) and anti-RBD IgG titers (mean: 941.3 ± 514 IU/mL) except for one that had low anti-S IgG (index: 2.72) and no detectable anti-RBD IgG. Vaccine-induced neutralization was high for all (mean inhibition: 89.68 ± 13.97 %) except for the same subject with low anti-S IgG. RBD specific B-cells were detected in nine participants at a precursor frequency of 0.08 ± 0.07 %. The level of circulating RBD specific B-cells showed moderate correlation with anti-S and anti-RBD IgG (R² = 0.4371, Spearman r = 0.9121 and R² = 0.6353, Spearman r = 0.8619 respectively) and neutralization efficacy (R² = 0.3351, Spearman r = 0.8619). Upon bnt162b2 vaccination, virtually all subjects in this study display induction of functionally neutralizing antibodies. However, only one in three subjects have circulating RBD-specific B cells, the latter only showing moderate correlation with serology and functionality.