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Abstract Bachelor Project 2017-2018 (Pathology): Validation of the Autostainer Link 48

The aim of this research is to validate a new Autostainer Link 48 from the firm Agilent as replacement for the old platform. The aim of this research is to secure the correct sample staining before analysing analyse patient samples. The Autostainer Link 48 stains paraffin slides using immunohistochemistry. For this study, four parameters being tested: repeatability, reproducibility, correctness, homogeneity.

To test the repeatability, antibodies such as CD7, CK7, CD45, CK PAN, Ki67 being stained all together in one run, on three several days. There is one slide of every antibody in the run. That’s different with the reproducibility, there are three slides of every antibody being stained in one run. The antibodies being stained for reproducibility are the same as the ones stained for repeatability. Reproducibility is performed on one day, not on several days.

The correctness is examined by staining with antibodies such as PMS2, MLH1, MSH2, MSH6, CD117, S100, PDL-1, P53, BCL6 one time in a run. Last but not least there is the homogeneity as an examined parameter. Therefor, an antibody such as CK PAN or vimentin is placed in the staining platform on all 48 sites, to check if there is a homogeneous staining.

As a result of the study the four parameters were approved by the pathologists. At the end of the study, because all parameters were approved, the Autostainer was released for analysing patient samples. The validation process was carried out before the old Autostainer was removed.

Abstract Bachelor Project 2017-2018 (Ardolab, Clinical lab)Implementation of Point of Care Testing for Activated Clotting Time

AZ Delta decided last year to replace their current Activated clotting time (ACT) analyzers “ACTPLus by Medtronic” with the “i-STAT Alinity Analyzer by Abbott”. The main reason for this was that the eight analyzers of the year 2006 and one analyzer of the year 2004 were due for renewal.

ACT is determined to know how much heparin should be added during an operation. The ACT of a healthy person is 90 to 130 seconds without the addition of anticoagulant. During heart operations, the target ACT value is 450 seconds and more. This is a very large range, however there is no 'golden standard' ACT method, therefore, there is no “true” ACT value.

The aim of the experiments is mainly to test the i-STAT Alinity of Abbott by means of performance testing to decide whether the device can be used in the work field.

All nine instruments were tested using two control levels, the results were within the reference values of the manufacturer. The measurement variance was less than 5%. The reproducibility was tested on three instruments using control levels and a plasma pool, % CV was less than 5%. The normal reference values ​​were also determined and these comply with the range recommended by i-STAT. The correlation test confirms a good correlation between 2 devices.

From the results of the performance tests it can be concluded that the i-STAT Alinity Analyzer meets the criteria that has been defined by AZ Delta.

Abstract traineeship advanced bachelor of bioinformatics 2017-2018Implementation of mSINGS python script for assessment of microsatellite instability in colon tumors

Background and aim: Standard molecular diagnostic testing for metastatic colorectal cancers (CRC) includes analysis of somatic mutations in KRAS, NRAS and BRAF and assessment of ERBB2 amplification. More recently routine testing was extended with detection of microsatellite instability (MSI). Microsatellites are repetitive DNA tracts that are prone to polymerase slippage events during DNA replication. In healthy cells, such DNA replication errors are corrected by the DNA mismatch repair system (MMR). Loss-of-function of MMR pathway proteins (MSH2, MLH1, PMS1, PMS2, MSH6, or MSH3) results in variations in the repeat lengths, or microsatellite instability (MSI). MSI is the hallmark of Consensus Molecular Subtype 1 (CMS1) CRC subtype, encountered in 15-20% of all CRC: due to their DNA instability, these tumours are hypermutated and highly immunogenic, and tend to respond favourably to immune checkpoint inhibitor therapy.  Currently, MSI is measured by a separate test, either by PCR analysis of specific loci (MSI-PCR), or by immunohistochemistry staining for loss of MMR protein expression (MSI-IHC). To improve the efficiency of the diagnostic workflow, we aimed to integrate MSI testing in our standard NGS workflow.

Methods: mSINGS (MSI by NGS) is a python-based open source script for MSI analysis using NGS data. The script analyses 14 microsatellite loci, embedded in a hybridization capture-based gene panel (NimbleGen SeqCap EZ choice, Kappa Hyperplus workflow, Roche). The script compares the output of a VarScan readcount file of experimental samples to a baseline trained by microsatellite-stable (MSS) control samples. MSI status is determined by the fraction of unstable loci. We implemented mSINGS on a HPC (High-Performance Computing) cluster in PSB (Plant and System biology) Ghent, optimized visual display, automated the workflow and validated MSI-NGS to MSI-PCR and MSI-IHC as reference techniques

Results: First, mSINGS was tested on 3 MSS and 3 MSI colon samples, with concordant microsatellite status by MSI-PCR and MSI-IHC. After the recommended baseline validation for custom assays, 3 target genes were excluded to improve discriminatory statistical power. Results were visualized in R markdown and compared to IHC staining and PCR-based MSI measurement. Next, we tested mSINGS in a larger cohort of samples (n=30), using MSI-IHC as reference. Overall, MSI-NGS, using 11 discriminant biomarker regions, showed > 95% concordance with the reference assay thus validating its clinical use.

Conclusion: Implementation of mSINGS to analyse MSI status from available NGS data increases the efficiency of molecular classification in CRC tumours and provides a robust and accurate clinical tool to select patients potentially responsive to immunotherapy.

Abstract bachelorproef 1 2016-2017Drawing up a risk analysis of the administrative flow in the pathology

In the laboratory of Pathology of the AZ Delta Campus Westlaan in Roeselare, there is research on tissues and body fluids. The laboratory can be divided into four compartments. First of all, you have the room where the tissue is cut into smaller pieces and then placed in cassettes. The next compartment of the laboratory is the room “cutting and coloring”, where the cassettes are embedded after treatment in the device. The next step is to cut the embedded tissues in sections. All sections are stained with hematoxylin-eosin staining or other additional colorings. Thirdly, there is the department of cytology where the body fluids are processed. And finally, the department of immunohistochemistry, that’s where the immunohistochemical stainings are performed.

The purpose of this bachelor test is to analyze the administrative part of this workflow with a risk analysis. What could all be wrong with the administration while processing a sample? What is the risk? How are these errors discovered? What could be the result of such a mistake?

For this purpose, a risk analysis was prepared using the FMEA, Failure Mode and Effect Analysis, method. FMEA is a risk analysis that requires a number of steps to minimize a mistake. In this risk analysis, the possible errors, causes, discovery and consequences were examined. Once these process steps were overrun, an RPN, Risk Priority Number, value could be drawn up. This RPN value looked at the probability of occurrence, the discovery and the consequence of this error. If this value was higher than six, an action was needed.

In the administrative workflow of the laboratory of pathology, many errors can occur. Most errors were observed when filling in the application form, registration and reporting. The biggest consequences are sample or patient change, which will make the RPN high.

The most common risks can be avoided through the establishment of an electric application form. By applying these measures, the margin of error will be minimized, making the RPN value less than six. These measures ensure that errors be corrected within the administration in the laboratory. This makes the risk of a wrong diagnosis smaller.

Abstract bachelorproef 2 2016-2017Comparison of three systems for immunohematological analyzes

The aims of this study were to evaluate the performance of a new automated system for immunohematological analyzes (Erytra® - DG Gel; Grifols) and to compare the data with two widely used systems, namely Ortho BioVue (AutoVue® - OCD) and DiaMed-ID (ID-Gelstation® – Bio-Rad). Blood group assays and antibody screenings are performed as pretransfusion tests and during pregnancy. This research was conducted in the context of a uniformization of the automatisation for immunohematological testing and preventive replacement of some older devices.

The evaluation and comparison of the three systems are performed over a period of five weeks. Most of the samples were collected from the routine. More special samples, for example with positive direct agglutination tests and positive antibody screening and identification, were gathered in the previous months or obtained from other laboratories. An analytical validation was performed, including a method comparison, sensitivity and reproducibility testing. Also operational functionalities were evaluated, such as turnaround time, volume testing, carry-over and error generation.

In general, it can be decided that small differences between the three methods were established on the basis of the method comparison. In the ABO assay, Grifols reacts more strongly to the reverse grouping and is more sensitive double populations. Weak Rhesus-D reactions were also picked up by Grifols.

In the screening of the indirect antiglobulin test (IAT) minor differences of sensitivity for certain samples were seen between the different methods. Grifols performed equal to Bio-Rad. In IAT identifications, Grifols is equal to OCD and Bio-Rad. In some cases, the enzyme phase was more susceptible to anti-Rh antibodies, while the Coombs screening proved less sensitive to anti-Lea. It should be kept in mind that these Grifols analyzes were done automatically and the other two methods manually. When samples are stored at refrigerator temperature, immunohematological tests can be tested reproductive up to seven days. When repeating weak reactions, Erytra®/Grifols proved less sensitive than other systems. The sensitivity assay with titrated anti-D also showed a weaker sensitivity to OCD. However, expressed in reaction strength, this was no more than one gradation. This may be due to the use of cards with glass beads instead of cards with gel.

At operational level, Grifols scored satisfactorily with the turnaround time determination, however, the BCSH directive for an ABO determination could not be achieved with any system. The volume tests show that Erytra® can produce a result with smaller volumes of whole blood with the exception of the IAT (required volume intermediate to ID-Gelstation® and Autovue®). The differences between the three methods were only minimal. None of the methods showed a sign of carry-over.

From this comparison, it can be concluded that the Grifols reagents are equivalent to Bio-Rad and OCD, depending on the test-defined differences, as noted above. The Erytra® device was appreciated as a robust system for the implementation of immunohematological analyzes. The comparison of the corresponding software was not included in this thesis. These results will be included in order to make a final choice, along with other aspects such as ease of use, finances, company service,... .

Abstract bachelorproef 3 2016-2017Serologische bepaling van de SOA pathogenen op dried blood spots
The purpose of this study is to detect hepatitis B, hepatitis C, HIV and syphilis in dried blood spots. The diagnosis of these sexual transmitted diseases is currently taking place on serum by detecting antigens and antibodies. Dried blood spots is an alternative way to carry out these analyses.                                                                                                Patients with known viral infections were tested. A few drops of blood collected through a fingerpick are collected on a DBS card. These cards are dried for 24 hours. When the drying process is complete, spots were punched out from each blood-soaked circle of the cards. These spots were eluted with PBS and were analysed for HBsAg, anti-HBc, HBsAs, anti-HCV, HIV and syphilis. The execution of this study consists of 2 parts. First, serology was tested whether it works on DBS. Then a method validation was performed. Negative EDTA blood was mixed with positive controls. The control material should be found. First, 50 samples were tested. 29 were found to be positive and 21 were found to be negative. The method validation was executed three times because negative results were obtained. During the first method validation, the samples were eluted too long. The method validation was executed again in the same way. There were still negative results. The third method validation was executed with a different volume of control material but some of the results were still negative.                                                                                    
The conclusion of this study is that serology on dried blood spots fails even though many literature proves the opposite. The exact reason for the failure could not be found because lack of time and a delivery problem with the DBS cards.
Abstract bachelorproef 2015-2016Validation FII mutation and factor V Leiden with GENEXPERT

The current study investigates the performance of GeneXpert for the detection of FII and FV Leiden mutation. Different aspects of the test including reproducibility, accuracy, sample type and sample storage conditions were compared to the currently used in-house developed PCR assay. In addition to these performance criteria, the total cost per test on both platforms was calculated.

An excellent performance of the GeneXpert assay was observed. Both reproducibility and accuracy were scored 100% compared to the in-house PCR. Moreover, an extended storage of the samples at 2-8 °C for 15 days as compared to the recommendations of the manufacturer had no impact on test accuracy. Despite this good performance and the ease of use of the GeneXpert assay it was decided not to implement this assay in routine practice. This decision was mainly based on the high cost of the geneXpert assay compared to the in-house PCR.

Samenvatting 1 eindwerk 2013-2014: Evaluatie van chromogene media (Brilliance (oxoid) en ChromID carba/OXA-48 (bioMérieux)) en Rapid CARB Screen (Rosco) voor een snelle detectie van Carbapenemase producerende Enterobacteriaceae (CPE)
CPE (carbapenemase producing Enterobacteriaceae) are bacteria that were first disco-vered in 2008 in Belgium in hospitals. These bacteria are resistant to carbapenems (antibiotics), making it so that only a limited number of antibiotics are remaining active against CPE. Infections by CPE often have a fatal ending. The distribution of CPE is not limited to hospitals. The genetic code to produce carbapenemase lies on the transposons or the plasmids, making the transfer of these genetic codes and so the spread of CPE quickly and efficiently (even to other species) even assuming endemic proportions. This makes it difficult to define a clear CPE screening group and it constitutes a great threat to public health.
The purpose of this research is to improve the detection of CPE in the clinical laboratory AZ Delta. The first improvement for the detection of CPE is to introduce an alternative to the Brilliance CRE agar namely ChromID Carba/OXA agar (bioMerieux) and use it if the results are good. As a second improvement one wants to introduce a new confirmation test namely the Rapid CARB Screen (Rosco). Finally, at the geriatric department of the different campuses of the hospitals in Roeselare one wants to take stool samples or rectal swabs of all the patients to screen for CPE in order to get an image of the number of pa-tients that carries CPE and for the performance of the plates at screening.
It may be concluded that the study population has no positive CPE carrier. However, this population does not fully reflect the true population. Therefore it can’t be said with certainty how many patients there are CPE positive in Roeselare-Menen region. The ChromID plate is a more specific and selective plate than the Brilliance CRE plate, but the plate is more expensive and few literature can be found on the sensitivity of the ChromID plate. The Rapid CARB Screen test is a quick and easy to implement confirmation test, but it has only a low test sensitivity and negative predictive value of 72.5 % and 69.2 % respectively. This makes it a less useable test in the routine.
Samenvatting 2 eindwerk 2013-2014: Validation of Pefakit APC resistance on STA-R Evolution
Activated protein C resistance is the most common hereditary defect associated with  thrombosis. It is caused by a disturbed inactivation of factor Va by activated protein C. This defect is usually due to a point mutation in the factor V gene, and results in the substitution of amino acid arginine by glutamine. The point mutation can be  homozygous and heterozygous.
The Pefakit® APC-R Factor V Leiden is a functional coagulation assay on citrated plasma. In contrast with the traditional APC resistance testing, the Pefakit APC-R test is based on the prothrombinase complex. When APC is added to the plasma of patients with APC resistance, the APC coagulation time is extended less strongly than normal plasma. The clotting time is measured, both with and without APC. In the case of a patient with APC resistance, the ratio will be smaller than in normal individuals. This kit also makes a distinction between homozygotes and heterozygotes.
The structure of this thesis starts with a theoretical background of hemostasis, coagulation inhibitors, thrombophilia and APC resistance. After that the application in the laboratory and the actual validation is described. The validation consists in determining the intra-run and inter-run reproducibility and in checking  the accuracy. In addition a method comparison with the Coatest® APC™ Resistance V was performed and the reference values were checked.
Pefakit APC-R is a high-performing test and both the reproducibility and the accuracy are good. Pefakit provides more information than the Coatest APC because there is on the one hand a clear separation between normal and deviating results, and on the other hand it makes a distinction between homozygotes and heterozygotes.


Ardooisesteenweg 276
8800 Roeselare


Traineeship supervisor
Dr. Anne Vandewiele
Traineeship supervisor
Dr. Inge Vanhaute
Traineeship supervisor
Eline Verhoye
Conny Van Keirsbulck
Steven Vervaeke
Traineeship supervisor
Geert Martens
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