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AZ Alma Eeklo

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Abstract bachelorproef 2016-2017: Validation of Hep-par 1 and Glypican 3 for immunohistochemical staining procedures
Immunohistochemistry (IHC) is one of the most commonly used research tools in anatomic pathology laboratories. It is widely used to purchase diagnostic and prognostic information and also to follow-up the treatments. To obtain optimal results of IHC tests, the laboratories must validate the immunohistochemistry assays and the antibodies, before those are placed into clinical service. Hepatocyte specific antigen and Glypican 3 are being used for detection of hepatocellular carcinoma (HCC) and for identification of metastases of HCC-origin in formalin-fixed, paraffin embedded (FFPE) tissue sections. Those antigen-markers can be detected by immunohistochemical stainings with the Hep-par 1 and Glypican 3 antibodies. The purpose of this study is to validate Hep-par 1 and Glypican 3 using the staining protocols of Nordic immunohistochemical Quality Control (NordiQC).

This validation procedure includes the IHC-staining of multiblocks, containing punch specimens of positive and negative control tissues. The accuracy of the staining was evaluated by comparing the obtained results to the results indicted by NordiQC. The precision of the test was also established by defining the inter-run and the intra-run precision. After the evaluation of the results of the multiblock stainings and after validation of both antibodies, several adjustments have been made to the NordiQC protocol. The purpose of this adjustments was to obtain the same staining results with use of less antibodies.

Both Hep-par 1 and Glypican 3 perform an optimal staining results of FFPE tissue sections with the NordiQC staining protocol and are validated. Changes to the original protocol did not delivered better results. 

 
Samenvatting eindwerk 2014-2015: Checking the reproducibility of the immuno-histochemical MSI screening
The purpose of this study is checking the reproducibility of the immunohistochemical microsatellite-instability (MSI) screening in intestinal tissue. MSI occurs in people with the Lynch syndrome, another name is hereditary non-polyposis colorectal cancer. Microsatellites are short DNA-sequences that frequently occur in the human genome. Patients who are diagnosed with the Lynch syndrome can have defects or instability in there genome.
The immunochemical MSI-screening tests the tissue with four antibodies, namely MSH2, MSH6, PMS2 and MLH1. It can show in which of the four antibodies there is a possible mutation.
The test is seen as an initial test for MSI, when this test indicates a positive result, another test at the genetic level will follow to determine a diagnosis. It is therefore important that this test is done, so that the genetic research can be carried out much faster and more focused.
There were eight samples tested which were tested previously in a different laboratory. All eight of the samples were treated with the four antibodies and reviewed by the pathologist. Afterwards the obtained results were compared with the already known reviews from the other laboratory.
The results of seven out of the eight samples were fully in line with the other results. All seven were considered negative. This means that the inspection of the nuclei is successful for all four antibodies and that there are no indications of mutations. One sample, as compared to the other laboratory, has a different result. The result differ only to a certain extent.
The divergent sample had been negatively rated. It was noted that the tissue treated with PMS2 showed a reduced colour. There is in fact generally known that this antibody significantly colours less intense than the other three antibodies. The other laboratory has interpreted it differently and decided to take this reduced staining as an argument for a mutation in PMS2.
The conclusion of this study is that the colouration obtained good results and the colouration itself does not show abnormal findings. Because the interpretation of the test is done by the pathologist much depends on his assessment.
 
Samenvatting eindwerk 2013-2014: Optimizing of an immunohistochemical staining with the PHH3 antibody
The Phosphohistone-H3 (PHH3) antibody is a primary antibody that binds to the PHH3 antigen. This binding can be detected by the aid of an enzyme linked to a secondary antibody. It causes a brown precipitate on the place where mitosis takes place. This staining makes tumor differentiation easier because it shows the cells who are in mitosis.
The PHH3 antigen refers to the H3 histone that will be phosphorylated during mitosis at serine 10 or 28 when the chromatin binds around the histones to form nucleosomes. This way, the chromatin will condensate to chromosomes.  
The aim of this bachelor thesis concerns the practical implementation of the immunohistochemical staining with the PHH3 antibody. Different protocols that vary in time of incubation with the PHH3 antibody and pretreatment steps will be compared. This research has shown that the protocol with an incubation time of 16 minutes and a pretreatment CC1 mild of 38 minutes has the best results. The protocols were each performed on a poorly differentiated invasive carcinoma of the breast, an invasive poorly differentiated ductal adenocarcinoma of the breast, a leiomyosarcoma located at the elbow and a malignant melanoma nodular type. The immunohistochemical staining was performed on the Benchmark XT from Ventana.
 
Samenvatting eindwerk 2011-2012: Immunohistochemie: Op punt stellen van dual stainings
Voor deze bachelorproef werden er twee dual stainings op punt gesteld. Bij een dual of multipel staining worden er twee of meerdere antigenen op één weefselcoupe gedetecteerd, waardoor men minder weefsel en tijd verliest en waarbij men makkelijker tot een diagnose kan komen. Voor beide dual stainings was er reeds een standaardprotocol dat gebruikt werd om beide kleuringen verder op punt te stellen.
Voor de immunologisch kleuring op punt te stellen, gebruiken we de Benchmark XT van Ventana.
Een eerste dual staining is de cocktail van twee antilichamen, anti-p63 en anti-34βe12, met anti-AMACR. Deze dual staining wordt in de diagnostiek gebruikt voor het opsporen van invasieve acinaire adenocarcinomen en High-grade Prostatic Intraepithelial Neoplasie (HPIN) in de prostaat. p63 en 34βe12 kleuren bruin, AMACR kleurt rood. Voor deze kleuring werden er drie verschillende incubatietijden voor de primaire antilichamen onderzocht, namelijk 16, 32 en 44 minuten. Door een vergelijking van deze resultaten werd de beste kleuring, namelijk 32 minuten, vastgesteld.
De tweede dual staining is anti-AE1AE3 met anti-S100. Deze dual staining kan in de diagnostiek gebruikt worden om gemakkelijker carcinomen met perineurale invasie op te sporen, bijvoorbeeld in het colon. AE1AE3 kleurt bruin, S100 kleurt rood. Voor deze kleuring werden er vier incubatietijden van de primaire antilichamen onderzocht, namelijk 16, 20, 28 en 32 minuten. De resultaten van deze kleuringen werden vergeleken en de beste kleuring, namelijk 20 minuten, werd vastgesteld.

Address

Moeie 18
9900 Eeklo
3293760679
Belgium

Contacts

Traineeship supervisor
Christophe Vandenabeele
Traineeship supervisor
Katrien Leybaert
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