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VIB-IRC-Molecular Cell Biology / Bioinformatics Core Facility
Vlaams Instituut voor de Zee
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AZ West (Sint Augustinus Veurne)

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Abstract Bachelor Project MLT 2020-2021: Evaluation of a Beckman & Coulter ‘Sensitive’ Oestradiol Reagens on the Unicel DxI 800 Immunoassay Analyzer

The measurement of serum 17β-Estradiol is essential to understand physiology, development and health of reproductive processes in both men and women. The determination of estradiol concentration in serum is a major contributing factor in the follow-up and treatment of infertility problems in women. The laboratory of az West currently determines estradiol on the Abbott Architect i1000SR immunoassay analyzer. Other fertility hormones are determined on the Beckman & Coulter Unicel DxI 800 analyzer.

In the past the laboratory direction already tried to consolidate all fertility hormone determinations on one immunoassay platform. Using two analyzers implies loss of time as the primary sample has to be moved from one analyzer to another, and it also necessitates quality control measurements and daily maintenance on an extra analyzer during weekends. Furthermore, the Abbott estradiol assay has limited linearity which makes frequent extra determinations on diluted specimens necessary. However, former estradiol kits from Beckman & Coulter proved not to be sensitive and accurate enough for assessment of estradiol (E2) in low concentrations, which also was obvious from external quality control programs of the Belgian government in the past.

The aim of this work is to evaluate this new Access Estradiol Assay on the Beckman & Coulter DxI 800, which Beckman & Coulter themselves indicate as “sensitive”. For the evaluation of the new testkit, a few performance features were evaluated: sensitivity (LoB, LoQ and LoD), precision (reproducibility and repeatability), total analytical error, uncertainty of measurement, method comparison, measurement range and linearity. Stability in refrigerator and in freezer was also determined.

In conclusion, the new method shows a very good total repeatability and is acceptable within the maximum admissible error as obtained from Sciensano. It also showed a good linearity over a wide range of dilutions. From the LoD value it can be expected that the assay offers improved measurement of low levels of estradiol.

 

Abstract Bachelor Project MLT 2018-2019: VALIDATION OF TEST 1 XDL FOR BLOOD SEDIMENTATION
The erythrocyte sedimentation rate (ESR) is a widely used screening test to detect inflammation in peripheral blood. The reference method for determination of the ESR is the Westergren method. Now, az West is using the Monitor V20 as an automatic device. It’s interesting for az West to compare the Test 1 XDL from Alifax with the Westergren method and the Monitor V20 because of the different measuring method. The Test 1 XDL uses a fresh EDTA tube from the haematology lab when analysing the ESR, so no extra blood collection is needed.
Measurements with the Westergren method, the Monitor V20 and the Test 1 XDL are conducted using the same routine samples. Based on the obtained results, the paired t-test shows a significant difference between the results. The correlation between the Test 1 XDL and the other two methods is lower than expected. When using the formula of Fabry the correlation increases, but it’s still not as expected. The correlation is best with the Passing-Bablok regression. The Bland-Altman analysis shows a positive systematic bias. Therefore, results with the Test 1 XDL are systematic higher than those with the Westergren method and the Monitor V20. The repeatability is tested by measuring samples four times in a period of four hours. The CV is lower than the limit of 10%. Changing setting on the device ensures better repeatability. In the future, all the samples should be executed with new settings to achieve better results. When comparing all the results, the Test 1 XDL is more sensitive to an increase in the ESR. For this reason, the Test 1 XDL will give more positive results compared to the Westergren method and the Monitor V20. If there is no increased sedimentation with the Test 1 XDL, the result with the Westergren method and the Monitor V20 will also be negative.
The Test 1 XDL is easy to use, it’s a fully automated analyser for the measurement of the ESR. The determination of ESR with the Test 1 XDL is reducing contact with potential biohazard. The workload in clinical laboratories decreases using EDTA-samples.
When comparing the different measuring methods, the results were not always as expected. The variables of the Westergren method have no influence on the Test 1 XDL. The device is easy to use and more samples can be analysed, but for az West a smaller device should be a better choice.
 
Abstract bachelorproef 2016-2017: Validation of the Urine Hybrid Analyzer FUS-2000 for semi-quantitative examination and sediment examination of urine

Urine may be a waste product, but it still contains a lot of information and says a bit more about what's going on in the body. Current methodology for the diagnosis of diseases in the urinary system includes microscopic examination of the urine formed elements and chemistry analysis. These methods are time-consuming. The FUS-2000 was purchased to solve this problem. The device is an in vitro diagnostic instrument, which enables chemistry analysis as well as urine formed element analysis and counting through one sampling. So the purpose is to validate the FUS-2000 and consider if the device is able to take over the current routine.

For the validation of the device, a few performance features were evaluated. These features are subdivided into precision (repeatability and reproducibility), trueness and method comparison.

For the evaluation of the repeatability, the positive control of the sediment was measured 10 times one after another. In order to assess the repeatability, the coefficient of variation was calculated. The results showed good test repeatability, with a coefficient of variation (CV) lower than the proposed 8%. This means that the repeatability is acceptable.

For the evaluation of the reproducibility and the trueness, the positive control of the sediment was measured on ten different days. As in the determination of repeatability, the coefficient of variation is calculated. The results of the reproducibility obtained a coefficient of variation also lower than 8% and ensures that the reproducibility is acceptable. The coefficient of variation for the trueness is beyond the limit of -8 and 8%.

This is because the positive control has no reference value, but limit values ​​in which the results should fall. Therefore, for the determination of the trueness, the average of these limit values ​​was taken. So if there is no exact reference value, the accuracy cannot be properly determined.

For the method comparison, the kappa coefficient was calculated. Kappa is a value to determine the agreement between methods and is developed to account the possibility that raters would guess.  Like other correlation statistics, the kappa coefficient can range from −1 to +1. A kappa of 0 means that the amount of agreement can be expected from random chance and a kappa value of 1 means perfect agreement between both raters. A concordance table of results is first drawn up before kappa is calculated.

When the FUS-2000 is compared to the microscopic method, the kappa generally showed a good agreement between both methods for white and red blood cells.

For the chemistry analysis, the FUS-2000 was compared to the MIDITRON® JUNIOR II. There was generally a good agreement between both methods. For the ketones a low kappa coefficient was obtained. This is because there are too many negative results and they appear all in the same category in the correlation table. That’s why the chance agreement is high. To solve this problem, more urine samples must be analyzed and compared to each other so the results are more dispersed.

As a general decision, it can be said that the FUS-2000 is useful for the diagnosis of diseases in the urinary system and is suitable for taking over the current daily routine.

Samenvatting eindwerk 2014-2015: Immunohistochemical staining on Benchmark XT (Ventana) versus Bond-Max (Leica)
Immunohistochemistry is an important tool in anatomical pathology for differential diagnoses which can’t be determined by routine analyses using reagents such as hematoxylin and eosin. It is used to search for tissue antigens that indicate infectious agents and specific cellular populations. In this thesis, two different immunohistochemical devices, specifically Benchmark XT (Ventana) and Bond-Max (Leica), are compared with regard to time efficiency. The ultimate goal is to find out which of these two devices requires the least work from the laboratory worker.
Benchmark XT is already present in the lab and is used daily. Leica has provided a demo-unit and antibodies to allow a comparison. Before the time measurements can be done, the antibodies need to be validated on the new machine. In total there are eleven antibodies to be used. Each of these has to be validated before the antibodies can be used for daily analyses. The most important factors for the best results are the antigen retrieval technique and the dilution factor. For antigen retrieval, heat (HIER) is most commonly used. Sporadically though, the use of enzymes (EIER) is advised. All steps needed for the validation of a new immunohistochemistry device are described in a SOP, present in the laboratory.
When all antibodies give the desired result the comparison between the two devices can be done. In total, three runs have been performed on both devices with samples from the same patient . Time usage is measured during each step of the process. Unfortunately, the staining results weren’t always as good as hoped for, because there was not enough time to validate the antibodies optimally. The location of the tissues on the glasses and the use of covertiles also had a negative impact on the results. Apart from that, the time measurement results were clear. In all three cases the time needed for correct usage of Bond-Max is a lot less than for Benchmark XT. Also the runtime, not included in the intervention time of laboratory worker, is increased from forty-four minutes to one full hour for Benchmark XT. The conclusion for all three cases is clear; the Bond-Max (Leica) is less time-consuming than the Benchmark XT (Ventana).
 
Samenvatting eindwerk 2013-2014: IHC-kleuring op Benchmark XT (Ventana) versus Autostainer Link 48 (Dako)
On the Pathological anatomy service in the AZ-Sint-Augustinus hospital at Veurne, two immunohistochemical staining devices were studied in order to compare the workability of the staining devices.
The necessity of immunohistochemical staining methods in anatomopathological research does increase because, due to the use of typing a tumor, it is possible to suggest an appropriate and targeted therapy for the patient. The antibodies used must be specific for the antigen. The ability to visualize a particular component in tissue, demands continuous perfecting of the technique and the associated equipment.
The aim of this research is comparing the workability of two staining devices: the current Benchmark XT of Ventana and the demo unit Autostainer Link 48 from Dako.
The determining factors are the analyze time, the quality of the staining, incubation capabilities of the primary antibodies and the catalogue value.
The antibodies that were selected for this short period research are; 34be12, CDX-2, ER, Her-2/neu, HMB-45, HP, Ki-67, Melan-A, P16, P53, P504S en PR.
The used test tissues are: skin, colon, appendix, tonsil, prostate and breast biopsies.
The workflow of both equipment and the quality of the preparations are tested and measured. On average, the proceeding time of the Autostainer Link 48 from Dako is nearly one hour shorter than the current Benchmark XT from Ventana. However, the Autostainer Link 48 produces less medical hazardous waste than the Benchmark XT.
The preparations which were colored on the Benchmark XT generally had less non-specific background staining than the Autostainer Link 48. There should be taken into account that the antibodies and protocols used on the Autostainer Link 48 could be further optimized, but this wasn’t possible in the short time period of the research.
 
Samenvatting eindwerk 2012-2013: Technieken voor het concentreren
van cytologisch staal in paraffineblokken
In het laboratorium Anatomo – Pathologie in het AZ Sint-Augustinus te Veurne zoekt men naar methodes voor het maken van celblokken. Het maken van celblokken houdt in dat er een concentratie van het scytologisch staal nodig is, daarna kan men de cellen inbedden in paraffine. Tenslotte kan de laborant van de celblokken coupes snijden zodat men verschillende kleuringen kan uitvoeren op de cellen. Om stalen te concentreren zijn er meerdere methodes, tot op heden maakt men gebruik van de agarmethode. De cellen brengt men eerst in agar zodat ze makkelijk kunnen worden ingebed. De gebruikte methode levert echter vaak celarme preparaten op. In dit eindwerk zoekt men naar andere technieken om celblokken te maken. Het onderzoek is nuttig voor de pathologen en de patiënt, door meer te concentreren kan men het aantal te onderzoeken cellen verhogen en de diagnostiek verbeteren.
Via een literatuurstudie wordt er gezocht naar courant gebruikte technieken voor het maken van celblokken. Tegelijk worden de universitaire centra Gent en Leuven gecontacteerd om na te gaan welke technieken zij dagdagelijks gebruiken. Enkele interessante technieken werden geselecteerd namelijk de Histogel methode, de behandeling met formol/azijnzuur, Cytofoam Core en Cytofoam Disk.
Alle methodes worden gekleurd met de standaard Hematoxyline-Eosine kleuring en ook immunohistochemische (IHC) kleuringen namelijk TTF-1 en Ki67. Men vergelijkt de resultaten, indien een bepaalde techniek superieur is aan de agarmethode, dan zal de nieuwe methode geïmplementeerd worden in het labo.
Na de studie is er niet één bepaalde methode die boven de andere uitspringt. De Histogel heeft een laag het concentratievermogen en de turmorale cellen worden IHC zwak aangekleurd. De Cytofoam Disk absorbeert weinig cellen, waardoor de cellen enkel aan de rand zichtbaar zijn. Formol/azijnzuur en Cytofoam Core zijn op vlak van concentratievermogen en IHC kleuring beter dan de agarmethode. De cellen liggen bij de formol/azijnzuur methode dichter bij elkaar en bij de Cytofoam Core dringen de cellen door tot in de kern van de foam. Deze technieken hebben geen nadelige effect op de IHC kleuring.
Het besluit van de studie is dat indien de Cytofoam Core op de markt is, men de methode opnieuw moet testen tijdens de staalname. De Formol/azijnzuur zorgt ervoor dat de cellen dichter bij elkaar komen te liggen. De andere technieken geven slechtere resultaten dan de agarmethode.
 
Samenvatting eindwerk 1 2011-2012: Expressie van epidermal growth factor receptor in niet-kleincellige longtumoren
Het doel van deze bachelorproef is het op punt stellen van het primair antilichaam EGFR (epidermal growth factor receptor) op de Benchmark XT in het labo anatomo pathologie in AZ Sint augustinus te Veurne. Door het vergelijken van gelijkaardige primaire antilichamen van verschillende firma’s.
Er zullen  2 verschillende klonen van het  antilichaam EGFR uitgetest worden. Deze klonen zijn door verschillende firma’s geproduceerd.  Na de validatietesten zullen de voor- en nadelen van deze twee antilichamen  vergeleken worden, het beste primair antilichaam dat wordt uitgekozen zal  verder worden gebruikt in het labo Anatomo Pathologie in Veurne.
In deze bachelorproef wordt er uitleg gegeven over: de algemene immunohistochemie, het primair antilichaam EGFR, de Benchmark XT, het verband tussen EGFR overexpressie, longtumoren en eventuele behandelingen. De resultaten van het op punt stellen van het primair antilichaam EGFR worden ook gegeven in dit eindwerk.
Na de validatie van het primair antilichaam EGFR, kan er worden besloten dat het antilichaam van de firma Dako de meeste voordelen biedt. Dit antilichaam is met succes op punt gesteld in het labo anatomo pathologie in AZSAV ( AZ Sint augustinus Veurne) en is klaar voor routinegebruik.
 
Samenvatting eindwerk 2 2011-2012: Implementatie van een reagens voor bepaling foliumzuur na herstandaardisatie
Foliumzuur in serum en rode bloedcellen worden bepaald om folaat deficiëntie vast te stellen. Veel laboratoria gebruiken daarvoor volautomatische bepalingen op basis van  folaat-bindende eiwitten. Het UK National External Quality Control Assessment Service vond bij een recente evaluatie onaanvaardbaar hoge variaties tussen de verschillende methoden, die deels verklaard worden door het ontbreken van  een internationaal aanvaarde standaard. Een WHO internationale standaard IS 03/178 werd daarom gecreëerd en geëvalueerd op basis van een serumpool van humaan serum.
In lijn hiermee heeft de firma Beckman Coulter zijn calibratoren, die gebruikt worden voor kalibratie van hun foliumzuur reagens, gestandaardiseerd ten opzichte van deze WHO Internationale Standaard 03/178.
Dit eindwerk beschrijft evaluatie en implementatie van de geoptimaliseerde methode voor folaat bepaling van Beckman Coulter op Unicell DxI 800, waarbij de calibratie met deze geherstandaardiseerde calibratoren uitgevoerd wordt.
De nieuwe testkit vertoont een aanvaardbare imprecisie (<10 %) zowel rond de cutoff waarde als in het hogere meetgebied. Door de herkalibratie ondergaan patiëntstalen wel een belangrijke upward shift voor serum folaat. Door gebruik van een ander cutoff leidt dit evenwel niet tot een verlaagde sensitiviteit en specifiteit voor het vaststellen van folaat deficiëntie.
Tags: anatomical pathology (mlt) clinical lab (mlt)

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Iepersesteenweg 100
8630 Veurne
058333111

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Traineeship supervisor
Nathalie De Wever
Traineeship supervisor
Annick Verhaeghe
Traineeship supervisor
Johan Gunst
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