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AZ Sint-Lucas Gent

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Abstract bachelorproef 2016-2017Validation of the Sysmex-XN body fluid mode for cell count and differential

The aim of this research is to evaluate the performance of the new body fluid module on the Sysmex-XN hematology analyzer (XN-BF) for blood cell count and differential in body fluids in the laboratory of AZ Sint-Lucas Ghent. Along with other validation parameters, a method comparison with manual microscopy was performed to evaluate the accuracy of the body fluid module.

Automated blood cell count and differential in body fluids add a great value to medical laboratories in comparison to the labor-intensive and time-consuming manual microscope method. The hematology analyzer needs to be validated according to the laboratories standards; therefore the XN-BF was evaluated according to these standards.

During a period of nine weeks, 110 samples (42 pleural fluids, 17 synovial fluids, 22 ascites, 2 continuous ambulatory peritoneal dialysis (CAPD) and 27 cerebrospinal fluids (CSF)) were used for method comparison between the XN-BF and manual microscopy for blood cell counting (Fuchs-Rosenthal counting chamber) and differential (cytospins). Further evaluation included inter-observer variation (manual method), precision, carry-over, linearity and Lower Limit of Quantitation (LLoQ).

Inter-observer variation for the manual counting method showed a coefficient of variation of 8,17% for total nucleated cell count (TC) and 4,66% for red blood cell count (RBC). For the method comparison in pleural fluids, good correlations (R2=0,972; R2=0,994) were found for TC and RBC >1000/µl counts, despite the bias found with the Passing and Bablok regression analysis in TC (y=80,7980+1,1919x). Excellent correlation for TC and RBC (R2>0,980) was found in synovial fluids, yet a constant bias was found again for TC (y=154,4702+0,9890x). In ascites and CAPD, good correlations are found as well (TC: R2=0,964; RBC: R2=0,989). However, the XN-BF systematically counted more TC compared to the manual microscopy (y=-0,03084+1,2419x). In CSF <10 TC/µl (n=21) no significant or constant bias was found, but correlations were worse compared to the other fluids (R2=0,817). Regardless, good correlations were found for RBC (n=25) and TC >5/µl (n=7). For the differential count, the XN-BF systematically counted less monocytes compared to the manual method in all fluids. Results of comparing the manual reporting for malignant cells with the High-Fluorescent fraction on the XN-BF showed that these cells are located in this region, but that the XN-BF is not specific enough for quantitation of these cells. Furthermore carry-over was negligible, precision was excellent (CV%: TC=2,3%; RBC=0,0%; neutrophils=6,4%; lymphocytes=4,2%; monocytes=5,1%), linearity was good for both RBC and TC (R2=0,99) and the LLoQ for TC was defined at 4/µl.

The XN-BF can be used in the laboratory for fast counting of TC and RBC, if nucleated cell counts <10/µl are counted again manually (CSF). Differentiation through microscopy is still needed due to the low correlations in monocyte differentiation and the lack of precision for reporting of malignant cells.

Samenvatting eindwerk 1 2014-2015: Optimalisatie ALK CISH voor een medisch laboratorium
About 3-4% of all NSCLC harbor the ALK-EML4 translocation, this seems not enough encouragement to detect every case of this ALK translocation, until Crizotinib was discovered to be a very effective treatment for ALK-EML4 positive NSCLC patients. Because these patients have a better prognosis than other NSCLC patients, this 3-4% is a very significant subset.
The standard procedure for the detection of ALK gene rearrangements until now consists of a screening with immunohistochemistry and confirmation using fluorescent in situ hybridization. Both techniques have their own advantages but also encounter some problems. With this in mind, the search for alternatives has already brought up a promising technique: chromogenic in situ hybridization.
In this work, a CISH-protocol was tested for the first time in the laboratory of pathology at AZ. Sint-Lucas Ghent. Whether or not this chromogenic test can or should be implemented in the laboratory as an alternative to FISH is the main question of this thesis.
An ALK CISH kit was bought from Zytovision. This kit consists out of the ZytoDot 2C CISH Implementation Kit and the ZytoDot 2C SPEC ALK Break Apart Probe and is used to detect ALK gene rearrangements by hybridizing a DIG –and DNP-labeled probe to the target tissue. Then the tissue is incubated with primary antibodies anti-DIG and anti-DNP. After washing away excess antibodies, the secondary antibodies, labeled with peroxidase (HRP) and alkaline phosphatase (AP), are bound to the primary antibodies. Finally, the HRP and AP are visualized with the supplied substrates AP-Red and HRP-Green.
The CISH was performed eight times, with four different types of tissue. Results were evaluated by Dr. De Potter and Koen Jacobs on three criteria: intensity of the signals, specificity and counterstaining. Normal lung tissue was used in three runs of which two failed. Tumor breast tissue gave good results in all six runs, tumor lung tissue only in one out of three. In the final two runs an ALK-EML4 control from Horizon was successfully stained.
Although the kit has proven its use, optimal results could not be achieved using this manual ALK CISH protocol because runs have to be performed under very strict conditions.
Generally, in a clinical lab, incubation temperatures cannot be monitored well enough to guarantee reliable results. Using specialized equipment, such as a Hybex incubator, this test could give better results. Even distribution of the antibodies on the tissue is another problem that could be solved with specialized equipment. Finally, performing ALK CISH in this manner takes too much hands-on-time to be able to implement it in a clinical lab.
 
Samenvatting eindwerk 2 2014-2015:
Validation of immunohistochemical staining by using multi blocks
The purpose of this study is the validation of a number of immunohistochemical staining that are performed in the pathology laboratory. This staining looks for specific antigens by using antibodies. On the base of this technique there can be determined whether the patient is a carrier of a tumor, a micro-organism, etc. For the interest of the patient it is important that these antibodies stain optimally the antigens, because based on these colorings, the treatment of the patient can be determined.
In this task, the antibodies CD5, CK pan, Cyclin D1, Ecad and PAX-5 will be optimized and validated. At first the accuracy of the staining will be determined. This by comparing the obtained result to the actual values that were created by NordiQC. When these conditions are met, then will be the precision determined by defining the inter-run precision.
For this research there are four control blocks created that are indicated by NordiQC. These consist of four different kinds of normal tissue that react with the antibodies in a specific way. For these five antibodies,only block one will be used. From this first block is a slide prepared for a Hematoxylin-eosin staining, this makes the tissues easy and quickly to evaluate. When the control block is found to be in order, another slide has been prepared for the IHC staining. After which the optimizing and validating can be started.
When the coloring fulfils the requirements it will be validated. However when the coloring is not satisfactory, the protocol will be changed. The incubation period of the primary antibody can be changed, but also the duration of the pretreatment, besides an option buffer can be added or passed on to another detection system. 
Four of the five antibodies are optimized and validated. For the three antibodies (CD5, cyclin D1 and PAX5), the original protocol conforms to the requirements established by NordiQC. Also with these three antibodies, the inter-run is optimal, in all runs is the same staining.
The fourth antibody, CK pan is optimized and validated after many adjustments and tests. When the run is being watched, a small variation was observed between the different results. But the resulting staining is more specific than the staining of the original protocol, which is chosen to implement the new protocol.
The fifth antibody Ecad has not improved after many tests, because there was not enough time. In the future these and the other antibodies will be optimized and validated by using the created validation blocks.
 
Samenvatting eindwerk 2013-2014:
Detection of HPV in head and neck cancer: Correlation between HPV-p16
 
Head and neck squamous cell carcinomas (HNSCC) are the 6th most common cancers worldwide. The most well-know etiological factors for HNSCC are smoking and alcohol use. Over the past 25 years is HPV, the necessary cause of cervical cancer, has also been etiologically linked with HNSCC. There are 120 types of HPV that are divided into Low Risk (LR) HPV and High Risk (HR-HPV). These HR-HPV has been linked to HNSCC. The current study evaluated a immunohistochemical correlation between HPV and p16, as a more cost-effective alternative to ISH and PCR detecting. P16 overexpression is not exclusive for a HPV infection.
The HPV viral oncogenes E6 and E7 are the main contributors to the development of HPV induced cancer. These oncogenes have the ability to bind host cell regulatory proteins, especially tumor suppressor gene products. The HPV oncoprotein E7 is known to bind inactivate retinoblastoma protein (pRB) which leads to upregulation of p16. P16 is a tumor suppressor protein that inhibits cyclin dependant kinases 4 or 6- binding to cyclin D which regulates the G1 cell cycle.
Paraffin-embedded, formalin-fixed pretreatment tumor tissues were available from the pathology archives. Tissues were immunohistochemically stained with the p16 and HPV antibodies. The results of HPV detection IHC was negative in all our samples, but 75% of the HNSCC showed a p16 overexpression. After IHC, 3 tissues are screened for HPV by PCR of every tumor type. 3 mouth-tumors, 3 tonsiltumors and 3 larynxtumors of which 2 were p16+ en 1 was p16-. The DNA piece coding for the L1 protein was amplified. Most of the results (93%) were positive for HPV-16.
L1 or the major capsid protein is one of eight known HPV specific proteins. It is produced within the cytoplasm and translocated into the nucleus. The L1 capsid is associated  with the minor capsid protein L2, which encapsulates the viral DNA to build new infectious viral particals. Once attached, the virions enters the host cell via L2 endocytose, the capsid become decraded, the virus DNA is released and routed into the nucleus of the cell. The virus genome then separately lies outside the chromosomal DNA of the host cell as a ringshaped episomal DNA molecule. This latent virus infection can only be detected with molecular biological methods.  The productive phase starts once differentiation of the host cell begins, the viral DNA starts to replicate to high copy numbers. The late proteins are synthesized, and encapsidated the viral DNA. Upon termination of the productive phase, the viral life cycle of the virus is completed without any malignant transformation. Only at that stage of the life cycle is the L1 capside protein detectable.
When the virus enters the host cell there is a primary infection, latent infection. In that stage is the DNA episomale. When it becomes a productive infection the DNA replicates and there is also an amplification of the DNA. In this stage there is a transformation of the host, the DNA is integrated in the genome of the host. Only in the termination of the productive phase there is a release of L1 proteins. When it comes to a premalignant transformation there is no release of L1 proteins. There is a transformation in all host cells without any virus production.
All the patient tissue had a tumor, they belong to a malignant transformation. This is the reason why all results are negative with IHC or maybe because IHC isn’t a very sensitive method.
83% of the results of PCR where positive for HPV-16. These numbers are consist with other studies showing a correlation of 93% and 86,7%.  
It is also found that 87,5% of HPV-16 were positive for p16 and 75% of HPV-16 are negative for p16. This shows that p16 is not a reliable marker for HPV in head and neck tumors.
 
Samenvatting eindwerk 2010-2011Kwantitatieve bepaling van hepatitis B DNA en hepatitis C RNA met behulp van het Rotor-Gene Real-Time PCR toestel
Infectie met hepatitis B (HBV) en hepatitis C (HCV) virussen zijn belangrijke oorzaken van chronische leverontsteking. Naast de detectie van HBV DNA en HCV RNA, zijn ook de bepaling van de virale lading van belang voor de therapie.
In het laboratorium Moleculaire Biologie worden de kwantitatieve bepalingen van HBV DNA en HCV RNA tweewekelijks uitgevoerd op serumstalen. Momenteel wordt hiervoor de COBAS® TaqMan® kit van Roche gebruikt. Deze assay is duur en kan enkel worden uitgevoerd op het Roche COBAS® TaqMan® 48 Real-Time PCR toestel. Voor alle andere real-time PCR analyses wordt binnen het labo gebruikt gemaakt van het Qiagen Rotor-Gene toestel. Hierdoor wordt de voorkeur gegeven om de HBV en HCV analyses in de toekomst over te zetten naar een test op het Rotor-Gene PCR toestel. Het doel van deze bachelorproef is het opzetten, valideren en implementeren van een HBV en HCV test op het Rotor-Gene Real-Time PCR toestel. Hiervoor worden de Qiagen Artus® HBV en HCV kits uitgetest. Deze zijn reeds gevalideerd voor gebruik met het Rotor-Gene toestel.
De validatie van de Qiagen Artus® HBV en HCV kits op het Qiagen Rotor-Gene toestel voor de real-time kwantificatie van HBV DNA en HCV RNA in serumstalen, uitgevoerd volgens de procedures, voldoet aan alle vooropgestelde vereisten. Daarnaast blijkt uit een prijsvergelijking dat de Rotor-Gene Q Artus® HBV/HCV Real-Time PCR test (Qiagen) goedkoper is. Enkele parameters moeten echter nog verder worden gevalideerd: de reproduceerbaarheid met behulp van de Rotor-Gene 6000, de detectielimiet van HCV en de keuze tussen de 36-well en de 72-well rotor van het Rotor-Gene systeem. Indien ook aan deze criteria is voldaan, kan deze test geïmplementeerd worden in het laboratorium Moleculaire Biologie.

Address

Groenebriel 1
9000 Gent
09/2246445
Belgium

Contacts

Traineeship supervisor
Henk Louagie
lab@azstlucas.be
Traineeship supervisor
Koen Jacobs
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