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AZ Groeninge

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Abstract Bachelor Project 2017-2018 (Clinical lab)Comparison and validation of reagents for analysis of trombophilia parameters on the Sysmex CS2100i

The continuous improvement of the quality of the tests used in a medical context is an important aspect of modern, clinical laboratories. One of the ways of achieving this objective is by continuously (re)evaluating and improving the reagents used in the lab. This study therefore aims to compare and validate different reagents for various thrombophilia tests currently performed in the laboratory of AZ groeninge. The main objective of this study is to obtain an effective and reliable test for the detection of protein C, protein S and antithrombin deficiency and for the screening of haemostasis anomalies like activated protein C resistance (APCr). These tests are helpful for an accurate and effective treatment of thrombophilia patients and therefore they should be examined and tested in detail. Multiple firms, namely Siemens, Hyphen Biomed and Werfen offer reagents for the measurement of these four parameters in citrate plasma samples. 
The validation of each reagent is achieved by determining the repeatability, reproducibility, bias and total error of each test. The various tests are also checked for the effect of possible interfering components like a highly haemolytic sample or the presence of the anaesthetic propofol in the citrate plasma. A method comparison is also performed between the different reagents. Finally a comparison is made of both the cost and shelf life of each reagent. All measurements are performed on the Sysmex CS2100i. After gathering the above mentioned data, the most optimal test will be recommended for usage in the laboratory of AZ groeninge.

The following results have been obtained:
The Werfen COAMATIC Protein C assay is the most recommended assay for the measurement of protein C levels in plasma. Both the total error and the precision have been determined to be the best of the three examined assays. In addition of this great performance, the shelf life of this test (three months at 2-8°C) is longer than the shelf life of any other examined reagent.

The INNOVANCE Free PS Ag assay of Siemens is the best choice for the evaluation of protein S. It shows great performance on the CS2100i and has other good characteristics like low cost and a decent shelf life.

For the detection of antithrombin deficiencies, the tests offered by Hyphen Biomed and Siemens are nearly equal in both performance and cost/shelf life.

Because of a continual calibration failure due to an unknown cause, the APCr assay of Hyphen has not been taken into account for the final choice of APCr test. The Siemens ProC Global test shows less false positive results and is less influenced by the presence of propofol in a patient’s blood. However, the performance of the ProC Ac R test is better than the ProC Global test. Therefore the laboratory itself should decide which of the two ProC assays has the most significant advantage when used in the lab.
Due to the limited availability of reagents and patient samples, the method comparison has only been performed on a low amount of samples. If possible the laboratory method comparison should be reperformed in the future on a higher amount of citrate plasma samples. It is also recommended to evaluate the reliability of the reference values and the cut off values mentioned in the leaflets of each company before applying them in the laboratory.  

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 bachelorproef 1 2016-2017Validation of procalcitonin analyses

Validation of procalcitonin analyses. The three kits (Vidas Brahms PCT, Elecsys Brahms PCT, and PCT LiquiColor Assay) were evaluated and compared to each other to choose the best method for routine use.

There is more and more interest in procalcitonin as a biomarker for sepsis due to its advantages against others conventional markers. Procalcitonin is a good marker for bacterial infection such as sepsis and that does not increase in case of viral infection. Furthermore, procalcitonin provides a fast diagnosis which is very important for sepsis patients.

The kits were compared on the basis of the repeatability, reproducibility and the trueness. Three concentration levels (negative, weakly positive and strongly positive) were measured ten times on the same day for the intra-run imprecision. For the inter-run imprecision, the three concentration levels were measured one time a day on ten different days. The intra-run and the inter-run of the methods have to meet the acceptation criterium of standard deviation <0,0375 ng/ml procalcitonin. Samples of sepsis patients, patients infected by influenza/RSV, patients on meropenem and intensive care patients were used for the trueness. The trueness of the methods was evaluated based on the absolute bias plot with an acceptation criterium of ± 0,075 ng/ml and on the passing-bablok with confidence intervals of 95%.

The repeatability and the reproducibility of Vidas Brahms PCT met the acceptation criterium for the three concentration levels. Only the negative and weakly positive concentration levels of Elecsys Brahms PCT met the criterium. For the PCT LiquiColor Assay, the weakly positive and strongly positive did not meet the criterium of the repeatability and the repeatability of the negative concentration level can not be measured because the results were below the lower limit of quantification. Therefore, no further research was being done for PCT LiquiColor. Vidas Brahms PCT and Elecsys Brahms PCT both did not meet the criterium for the trueness.

According to the results of the repeatability and reproducibility, it can be concluded that the Vidas Brahms PCT is better than the other two methods and suitable for use in the routine laboratory. However, the trueness of this method did not meet the criterium. The main reason for choosing the Vidas Brahms PCT is its lower price.

Abstract bachelorproef 2 2016-2017Comparative study of the EGFR test on the IdyllaTM

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Abstract bachelorproef 1 2015-2016Validation of a Phadia 250 system for the measurement of tryptase

Tryptase is an enzyme, made and stored in mast cells. The measurement of tryptase is a useful marker in the diagnosis of systemic mastocytosis, allergic reactions and anaphylaxis.

Tryptase can be measured with a Phadia system. These days AZ Groeninge sends the samples for measuring tryptase to UZ Leuven. But AZ Groeninge would like to investigate the possibility of determining tryptase in their own laboratory. So the purpose is to validate the tryptase assay on the Phadia 250 system in the laboratory of AZ Groeninge.

For the validation of the Phadia 250 system of AZ Groeninge, a few performance characteristics were evaluated: precision (reproducibility and repeatability), trueness, uncertainty of measurement, method comparison and measurement range. Also the stability of tryptase in serum was evaluated.

For the evaluation of the reproducibility and the trueness the tryptase level of the Tryptase Control and the Curve Control was measured on ten different days. The coefficient of variation (CV) and the relative bias (Brel) were calculated. The CV is used to evaluate the between-day reproducibility and the Brel to evaluate the trueness. The CV was lower than the proposed 10% and the Brel was situated between -10% and +10%.

Two patient samples, one with a high tryptase level and one with a low tryptase level, were used for the evaluation of the repeatability. The concentration of tryptase was measured ten times in the same run. The within-run precision was acceptable with a CV lower than 10%.

The uncertainty of measurement (Total Error (TE) = Brel + 1,65 * CV) was determined with the previous results. The TE of both controls was lower than the calculated error limit of 26,5%.

Since there was no reference method available, we compared results of 32 samples that were determined in UZ Leuven on a Phadia 1000 system. A Wilcoxon test showed a statistically significant bias. Passing-Bablok regression analysis showed a significant slope and a non-significant intercept. However, the difference was clinically acceptable according to the absolute difference plot between the two methods.

The manual of the Phadia 250 system states that the machine can measure tryptase levels between 1 and 200 µg/L. Calibrators were used as samples and the Brel was calculated. With a Brel between ±15% the measuring range between 1 and 200µg/L was confirmed.

Regarding stability tryptase should be measured within five days if a sample is stored in the refrigerator according to the manufacturer. Tryptase was measured on five new samples and then stored in the refrigerator. Tryptase was measured again after five and seven days. There was no statistically significant difference between the results from day one, five and seven. Thus samples can be stored for up to seven days in the refrigerator.

The conclusion is that the Phadia 250 system in AZ Groeninge can be used for the routine measurement of tryptase in serum. It showed acceptable performance characteristics regarding precision, trueness, measurement uncertainty, method comparison, measuring range and stability.

Abstract bachelorproef 2 2015-2016Development of controls for immunohistochemical staining

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Samenvatting eindwerk 1 2014-2015: Verification of ChromID CPS Elite
At the laboratory of microbiology AZ Groeninge in Kortrijk, urine cultures are analysed by ChromID CPS, a chromogenic media. A standardized urine sample is inoculated on the  culture medium. After incubation, when the growth can be interpreted by color and number, a plate count can be performed. In case of a significant growth there will be identification of the bacterial species by MALDI-TOF. This research compares ChromID CPS (CPS3) with a new formulation of the media, ChromID CPS Elite (CPSE). Both media are compared in a number of aspects like color, growth and Colony Forming Units. 200 random samples are inoculated on both media to determine the accuracy. There is also a quality research and test on shelf life in different conditions with QC-strains. At least some bacterial species are tested on selectivity and short incubation period on both media. The results of all tests are very equal with the product info.  The calculated accuracy is 97% which means a good agreement with CPS3. Some colors of the colonies are different from CPS3, that doesn’t make any difference for the identification.  It’s an advantage for the differentiation on the media. The medium can be read earlier because the growth after sixteen hours is already clear with most of the bacterial species. CPSE can replace CPS3 in the laboratory for the detection of urinary pathogens.
Samenvatting eindwerk 2 2014-2015: Opstellen van een immunoprotocol voor PTEN en IMP3
The subject of this thesis is to optimize two immunohistochemical stainings for IMP3 and PTEN. These are two proteins that are involved in a wide range of tumours. The two stainings are used to differentiate two subtypes of endometrial carcinoma: endometrioïd endometrial carcinoma (type 1) and endometrial papillary serous carcinoma (type 2).
The different subtypes of endometrial carcinoma have a different grade. There are high grade and low grade lesions, both having a different approach and treatment. That is why a good differentiation method is required. This method can be obtained by using IMP3 and PTEN. It is important to optimize the immunohistochemical staining protocol in order to achieve a clear difference between the two types of endometrial carcinoma.
Dako, the manufacturer of the antibodies that are used in the staining in this project, suggests some ideas for the staining protocol. In this thesis, some modifications were added to those suggestions. The test tissue is endometrial tissue with tumours, because it is known that IMP3 and PTEN have a different expression depending on the kind of tumour.
It turns out that the staining for IMP3 was clear and correct. The tumour cells that were supposed to have a brown colour, were indeed coloured. Importantly the negative tissue did not stain. The staining for PTEN however was not that successful as the background environment was also coloured brown. Moreover the tumour cells that were ought to be positive were only very lightly coloured compared to the background.
Although the background environment coloured brown for the PTEN staining, it was decided to keep the immunoprotocols the way they are now.
Samenvatting eindwerk 2013-2014: development of an antibody staining procedure for napsin A en anti-c-MYC on the BenchMark GX
The aim of this bachelor thesis is to develop and fine-tune two different immunohistochemical staining procedures who will be added in the routine colour panel. The staining procedures are based on the antibodies napsin A and anti-c-MYC, both used in the research and prognosis for various cancers.
The staining procedures are performed on the BenchMark GX of Ventana (Roche), this device can be used for both immunohistochemistry and in situ hybridization. Primary immunohistochemistry makes use of cell or tissue self-antigens that are detected with primary antibodies using the key-lock principle. Secondary the antigens are visualized with secondary antibodies using the DAB-chromogen method.
Napsin A is a polyclonal antibody purified from rabbit anti-serum. It is directed against a protein which is located in the epithelial cells from the lung and kidney. The test staining procedures carried out in the lab are on lung tissue. Napsin A is used for detection of adenocarcinomas and makes a difference between a primary or metastatic cancer stadium. Both have a different prognosis, so the therapy should be adjusted according the stage of the cancer.
Anti-c-MYC is a monoclonal antibody purified from rabbit anti-serum. It is directed against the N-terminus of a particular transcription factor. Anti-c-MYC is primarily used for detection of various lymphomas, including Burkitt lymphoma. The test staining procedures carried out in the lab are on lymph node tissue. The expression of the antigen can be correlated with the presence of a translocation between chromosome 8 and 14. If both are present, a particular therapy can be started. Overexpression on the other hand does not always mean that there is presence of a translocation.
The preparatory process of making tissue slides is performed by the lab technician. The slides can be labelled and the BenchMark GX carries out the process from deparafinization to the staining itself. The dewatering, assembling and distribution must be done by the technician before the doctor can examine the tissue. The first step to create an optimal test field for the process is a major maintenance on the equipment, which must guarantee that abnormalities in the staining are not made by the device.
The optimization of the protocol occurred in three phases. The first phase is to follow the protocol set under the rules of Ventana. The little information for napsin A acquired the starting protocol of anti-c-MYC. These results show that the staining is both macroscopic and microscopic dark, the brown DAB colour and the blue counterstain are both very evident. The second phase is to add two steps in the procedure, an extra antibody incubation and an extra wash. These will ensure that there will be no rests of reagents or colouring, this makes the staining lighter. Yet the blue background remains a little darker, so the counterstain is shortened in the third trial. The both colours now have the same intensity, so the protocol can be set and approved for the routine.
Samenvatting eindwerk 2012-2013: Evaluatie van de Euroflow "plasma cell dyscrasia tube" voor flowcytometrische diagnostiek van plasma cell myeloom
Plasmocyten zijn antilichaamproducerende cellen. Ze hebben een typisch uitzicht met een basofiele kleur en een ronde excentrisch gelegen kern.
Als plasmacellen ontaarden, kunnen er verschillende pathologieën ontstaan, zoals plasmacelmyeloom, monoclonal gammopathy of undetermined significance, monoclonal light chain deposition disease en solitair plasmocytoom.
Bij de diagnostiek van deze aandoeningen kan onder andere gebruik worden gemaakt van een flowcytometer. Hierbij worden de cellen één voor één gekarakteriseerd op basis van grootte, complexiteit en de aanwezigheid van typische antigenen op het celoppervlak.
Het Euroflow consortium heeft ervoor gezorgd dat verschillende flowcytometrische analyses gestandaardiseerd worden. In dit eindwerk is het de bedoeling om een door Euroflow voorgestelde test te valideren. De Plasma Cell Dyscrasie screening tube (PCD screening tube) zal de ‘in house’ plasmocytentube vervangen. Deze tubes kunnen plasmacelaandoeningen aantonen. De PCD screening tube bevat dezelfde merkers als de ‘in house’ tube, samen met enkele nieuwe merkers die extra informatie geven over de aandoening.
De nieuwe PCD screening tube werd geëvalueerd, door bij verschillende stalen de oude en nieuwe methode met elkaar te vergelijken.
Wanneer de merkers die in beide tubes voorkomen statistisch met elkaar vergeleken worden, kan worden besloten dat er geen significant verschil is tussen de ‘in house’ plasmocytentube en de PCD screening tube.
Ook zorgen de nieuwe merkers voor aanvullende informatie bij de diagnose.
De nieuwe PCD screening tube is dus zeker een aanwinst bij het diagnosticeren van plasmacelaandoeningen.
Samenvatting eindwerk 2010-2011:  Methode validatie van IllumigeneTM LAMP test voor de detectie van toxineproducerende Clostridium difficile in feces
“Clostridium difficile Associated Diarrhea” is een gastro-intestinale infectie die veroorzaakt wordt door toxines geproduceerd door Clostridium difficile (C. difficile). Aangezien deze infectie geassocieerd is met een hoge morbiditeit en mortaliteit, is een snelle en correcte diagnose van cruciaal belang voor een efficiënte behandeling.
In dit eindwerk wordt de C. Diff Quik Chek Complete van Techlab®, een snelle enzyme immunoassay die tegenwoordig gebruikt wordt in het AZ Groeninge voor het opsporen van C. difficile antigen en C. difficile toxines, vergeleken met een mogelijk meer gevoelige “Loop Mediated Isothermal Amplification assay” (IlumigeneTM van Meridian®).
Een prospectieve studie waarin tachtig stalen met zowel de C. Diff Quik Chek Complete (+ eventueel toxinogene cultuur) als de IllumigeneTM getest worden, toont aan dat de IllumigeneTM een iets betere sensitiviteit heeft ten opzichte van de C. Diff Quik Chek Complete.
Het tweede luik van deze bachelorproef, waarin selectief stalen opgenomen zijn die met de C. Diff Quik Chek Complete C. difficile antigen positief en toxine positief of negatief zijn, toont aan dat de IllumigeneTM een superieure sensitiviteit heeft ten opzichte van de C. Diff Quik Chek Complete.
Aan de hand van deze resultaten en een korte studie over de economische belasting en de “turn around time” van beide testen, wordt een driestaps algoritme voorgesteld waarin in eerste instantie gescreend wordt met de C. Diff Quik Chek Complete (+ eventueel toxinogene cultuur), maar de IllumigeneTM als supplementaire test gebruikt wordt indien het resultaat van de C. Diff Quik Chek Complete antigen positief en toxine negatief is. Zo kunnen 37,5% meer positieve stalen opgespoord worden zonder een forse stijging van de kostprijs.
Samenvatting eindwerk 2010-2011: Vergelijkende studie van immunohistochemische kleuringen
Het doel van deze bachelorproef is het vergelijken van immunohistochemische kleuringen. Specifiek worden enerzijds twee antilichamen van verschillende firma’s vergeleken. Anderzijds wordt een vergelijkende studie gemaakt tussen twee voorbehandelingen van dezelfde firma en dit voor drie antilichamen.
Het eerste antilichaam dat behandeld wordt in deze bachelorproef is het antilichaam tegen p63. Dit antilichaam werd oorspronkelijk aangekocht bij de firma Dako. Wegens het niet langer produceren van het p63 antilichaam moet er overgeschakeld worden naar een andere firma. De dienst pathologische anatomie heeft hierbij gekozen voor de firma Ventana. Door een vergelijkende studie te maken tussen het antilichaam tegen p63 van Dako enerzijds en van Ventana anderzijds, kan er besloten worden of het antilichaam tegen p63 verdeeld door Ventana een goed alternatief is, ter vervanging van het p63 antilichaam van Dako.
Bij de kleuring met het antilichaam tegen MSH6 van Ventana, blijkt de intensiteit van de aankleuring niet aan de normen te voldoen. Om te weten te komen of dit probleem ligt bij het antilichaam van Ventana, wordt besloten deze kleuring te vergelijken met een zelfde antilichaam van een andere firma. In dit geval kiest de dienst voor de firma Leica.
Voor de kleuringen met de antilichamen tegen HER2, ER en PR, wordt gebruik gemaakt van specifieke HercepTestTM en ER/PR pharmDxTM kits. Deze kits bevatten alle reagentia die nodig zijn voor de voorbehandeling en de kleuring van de coupes. De kits bevatten eveneens controlecoupes. De resultaten bekomen met behulp van deze kits, voldoen niet volledig aan de NordiQC normen. Aangezien deze kits gebruik maken van afzonderlijke voorbehandelingen, worden deze vergeleken met de standaard FLEX voorbehandelingen.
Uit de resultaten van de kleuring met het antilichaam tegen p63 van Dako en Ventana, kan besloten worden dat deze van Ventana een vergelijkbaar, tot zelfs beter resultaat oplevert.
De kleuringen met het antilichaam tegen MSH6 van Ventana en Leica geven een afwisselend resultaat. Daardoor is het niet mogelijk om een definitieve conclusie te trekken in verband met deze kleuring. Er moeten dus nog verdere onderzoeken uitgevoerd worden, om tot een beter resultaat te komen.
Voor HER2 zijn de resultaten van de FLEX procedure vergelijkbaar met deze van de HercepTestTM kit. De voorbehandeling via de FLEX procedure, blijkt dus geen betere invloed te hebben op de intensiteit van de aankleuring. Voor ER blijkt de FLEX voorbehandeling echter een beter alternatief. De aankleuring is algemeen meer aanwezig en is intensiever. Bij PR is dit resultaat zelfs nog duidelijker. Voor de ER en PR testen kan dus besloten worden dat de FLEX procedure zeker een beter alternatief is dan de afzonderlijke kits.
Ondanks de betere resultaten van de FLEX procedure, blijkt de intensiteit nog steeds niet te voldoen aan de NordiQC normen. Er moeten dus nog verdere onderzoeken gebeuren in verband met de intensiteit van de aankleuringen.


diverse adressen
8500 Kortrijk


Traineeship supervisor
Dorine Gadeyne
Traineeship supervisor
Dr. Alain Vanneste
Traineeship supervisor
Ann Dusselier
Traineeship supervisor
Olivier Heylen
Annelies De Bel
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