A.Z. Sint-Jan Brugge-Oostende AV campus Sint-Jan
For my internship I was asked to implement a web interface for monitoring quality parameters of Illumina runs. The data of these runs could already be visualized by the free application Illumina sequencing analysis viewer. This application has one major downside, it is not possible to compare values for the quality parameters between the different runs and samples. Being able to perform this on the web interface was the goal.
We worked in R to extract and read all the data for the runs. Using the packages XML and SavR these files could be handled in an R-script. SavR is used to parse and analyse Illumina SAV files, these files contain the quality parameters for one run. In the XML files, general info about the run can be found, such as run number and date. For some parameters extra steps were needed; calculating averages or discarding 0-values for example.
After processing all this data and calculating the needed values, these values are stored in a MySQL database thru the R-script. In the database we make a distinction between runparameters and sampleparameters. For the runs there are 23 different parameters in the database: RunID, Name, Total Reads, PF Reads, PF Percentage, Reads Identified, Q30, Density, PhasingR1, PhasingR2, PhasingR3, PhasingR4, PrephasingR1, PrephasingR2, PrephasingR3, PrephasingR4, Read1Length, Read2Length, Read3Length, Read4Length, Tiles, Date and Instrument. For comparing the samples only 4 parameters are used: RunID, Name, Fraction PF Reads and Reads.
Now that a database is created with all the data, we can call for these values, representing each run, to create plots and make it easy to compare different runs. For each parameter a separate script was created. In general, these scripts call for the info for each run in a loop for the corresponding parameter. After that a plot is made with this data and saved as an image. These images will be used in the web-interface.
The web interface contains 4 different start pages. The first one contains all info of the most recent run and comparison with the 20 previous runs. The second one is a list with all run numbers where the most recent one is on top off the page. The third one is the same but for samples. The last page is a list of instruments used between all the runs.
When clicking a run or sample on these pages, you get redirected to a details page for this particular run or sample. On this details page you can find a table with the values for all parameters. Underneath the table, you can find the comparison between the 20 previous samples/runs and the selected sample/run in the form of different graphs. Some graphs have cut-offs where a colour code can be applied. When clicking on an instrument, a list of run numbers is given. All these pages are linked together.
In the end we created a clear and simple web interface for comparing runs and samples. In addition an instrument page was created and the latest run has its own page. The script also runs automatically when a new folder with data is added to the parent folder.
The current method of sterility tests at the pharmacy and tissue bank of the AZ Sint-Jan Hospital in Bruges shows a number of shortcomings such as the fact that it’s very labor-intensive. These sterility tests are used to verify the absence of viable and actively multiplying micro-organisms in preparations. This is done to prevent intravenous infections.
The plan is to replace the current manual method with an automated method using the BACTECTMFX Blood Culture System.
In this research, different pharmacy preparations (smoflipid/vitalipid, norepinephrine, growth 92 without trace elements and PCA epidural), stem cells and bone product are spiked with different types of micro-organisms. These suspensions are injected into a hemoculture bottle with a sterile syringe and needle. The hemoculture bottle is incubated in the BACTEC system until it’s flagged as positive which means that it can adequately detect growth. After this, micro-organisms are identified in the positive bottles. This identification is done with microscopy (Gram stain), culture and massaspectrometry (MALDI-tof).
In the first project, the bone and tissue bank project, the BACTEC method is compared to the standard plating method. This comparison led to the conclusion that the BACTEC method is faster than the plating method (2.56 vs 11.18 days). The BACTEC system is also the most sensitive and therefore the most accurate to identify C.albicans (53.85 vs 15.38%), as well as A.fumigatus (46.15 vs 38.46%) and Fusarium spp. (38.46 vs 0.00%). For the second project, with pharmacy preparations, the BACTEC system was validated in this study. The BACTEC method was found to be a sensitive method for different types of micro-organisms: S.aureus (100.00%), B.cereus (87.50%), P.aeruginosa (100.00%), C.albicans (87.50%) and A.fumigatus (75.00%). However it appeared to be less sensitive for anaerobes (C.perfringens 43.75% and B.fragilis 50.00%). This is not due to the BACTEC system but is caused by coming into contact with oxygen when making dilutions.
It can be concluded that the BACTEC system is a much easier and faster method with less hands on time, than the classic plating method when it’s used with suspensions that include bone product or stem cells. The method is very sensitive with pharmacy preparations as well. The current manual method can therefore be replaced by the automatic BACTEC method in the pharmacy and tissue bank of the AZ Sint-Jan Hospital in Bruges.
The purpose of this research is to evaluate a new device (the MediMachine II) for automated tissue dissociation for flow cytometric analyses.
The current method for automated tissue dissociation is the GentleMacs Dissociator. Although already in use for several years, two main problems do occur. Sometimes an extra population is seen after an analysis, but there is no explanation for which cells this population indicates. The second problem is that sometimes malignant populations cannot be found with flow cytometric analysis. For these reasons, an alternative device will be evaluated (the MediMachine II). This device could be a solution for avoiding the two problems with the GentleMacs Dissociator.
To evaluate the device some parameters are examined. These are the correlation and difference in viability. These parameters will also be carried out with the manual method because it is known that with this method the least cells are destroyed. Also, the imprecision and the shelf life of a sample will be examined an as additional tests a price comparison will be made and the difference in ease of use will be examined. These tests will be done on lymph nodes and will be analyzed with the BD FacsCanto II flowcytometer.
The results of the tests show that the MediMachine II had higher correlation results for the analysis of CD19+. This can be important for analyzing B-cell chronic lymphoproliferative diseases. The difference in viability of the cells between the devices is notable. The stopping-gate of 5000 lymphocytes is more achieved with the MediMachine II than with the GentleMacs Dissociator. This may be due to the mechanical processing with the GentleMacs Dissociator, causing more destroyed cells. The imprecision is good with only a few values outside the limits. This is due to the heterogenicity of the tissue. Analyzing a lymph node is best within 24 hours. After that, the results can no longer be considered reliable. From the previous tests the MediMachine II is better but looking at the price and the ease of use, the GentleMacs Dissociator is better. The MediMachine II is more expensive and requires a manual and automatic processing step.
The results of the evaluation show that the viability of the cells is better with the MediMachine II and the manual method. This may be the reason that maligned populations with the GentleMacs Dissociator are more difficult to perceive. This parameter is an advantage for the MediMachine II. The switch to the MediMachine II will also depend on the other parameters because it’s more expensive and less easy to use.
Lynch syndrome is an autosomal hereditary disorder that is the result of a mutation in one of the MMR-proteins (MLH1, PMS2, MSH2 and MSH6). Patients with Lynch syndrome have 25 to 70% chance to developing colon cancer. The aim of this research is to evaluate the correlation between micro satellite instability (MSI) and immunohistochemistry (IHC). There is also an evaluation of the reliability of a two- antibody panel.
MMR deficiency can be detected by immunohistochemical staining and / or MSI analysis. This study is about immunohistochemical staining. These stains are performed on the MSI panel of the Roche Ventana device. The indirect detection method is the method most frequently used on the Ventana.
There is a 98,37% correlation between MSI and IHC. There is 0,00% possibility of false positive results and a 1,63% possibility of false negative results. When there are ‘possible’ results, these tissue sections should be checked for any misinterpretation of the IHC because these tissue sections show abnormal expression. The Shia et al study confirmed that a two-antibody panel is as reliable as a four-antibody panel. Isolated loss of PMS2 and MSH6 gives a greater indication of Lynch syndrome than MLH1 and MSH2.
There is a good correlation between MSI and IHC. Both techniques will continue to be applied at AZ Sint-Jan in Bruges. From this study there can be concluded that the IHC is reliable if an MSI analysis confirms colorectal carcinomas. The four-antibody panel is still used. In the future, a two-antibody panel can be introduced in the lab.
Punching was performed from FFPE tissue from oropharyngeal tumors. The DNA was extracted using the MagCore, then diluted and placed on the Cobas. The results were compared with those obtained from the p16 IHC and HPV ISH, tested on the Benchmark Ultra from Ventana (Roche). Two methods for measuring DNA concentration were compared, showing that the Qubit is more reliable than the NanoDrop. By comparing different diluents, it was decided that the extract should be diluted in 1 ml of preservcyt or 1 ml of 50% ethanol. It was tested on two samples whether the extraction step with the MagCore could be omitted and could be extracted manually instead, but this must be further tested with more samples. The quality of the DNA was tested on the basis of a QuantiMIZE run. This test showed that the DNA from the oropharynx was damaged or too fragmented. Discrepant results were obtained for two of the ten HPV HR+ samples. Two of the ten HPV HR+ samples gave an invalid value. Some of the other 6 samples were positive for HPR16, but invalid for other HR HPV and HPV18. Invalid results were all obtained for the four oropharynx LR+ samples. The invalid values can be explained by the fragmentation of the DNA.
Staphylococcus aureus is an important pathogen that can induce several types of bacterial infections. Over the past decades, there has been an alarming increase in methicillin-resistant Staphylococcus aureus (MRSA) strains.The aim of this study is to reduce the turnaround time (TAT) of the five-day MRSA screening in the AZ Sint-Jan Bruges hospital by using the Alere™ PBP2a Culture Colony Test, an immunochromatographic assay for rapid detection of methicillin resistance in bacterial colonies on culture media.
Over the course of eleven weeks, the test was performed on suspicious bacterial colonies from Oxoid™ Brilliance MRSA 2 media inoculated with patient samples. The results of the test were compared to the antimicrobial susceptibility test (AST) measured by the BD™ Phoenix™ M50. Additionally, the TAT after the introduction of this test was compared to the TAT of standard culture and AST screening.
Out of the 50 tested samples, 47 samples tested positive and three samples tested negative. One sample tested falsely positive compared to the AST. This resulted in a sensitivity of 100 % and a positive predictive value (PPV) of 97,87 %. Because of the selective components in the Brilliance MRSA 2 media resulting in only three negative test results, calculating the specificity and negative predictive value was not meaningful. The internal positive control of the test was positive for every test performed. Using the test resulted in an average TAT reduction of approximately one and a half days.
With a sensitivity of 100 % and a PPV of 97,87 %, the Alere™ PBP2a Culture Colony Test is suited for testing bacterial colonies from Oxoid™ Brilliance MRSA 2 media, resulting in an average TAT reduction of approximately one and a half days. After this study, use of the Alere™ PBP2a Culture Colony Test was implemented in MRSA screening in the AZ Sint-Jan Bruges hospital. In a future study, the impact of the TAT reduction on MRSA spread in the hospital could be investigated. Additionally, there could be experimented with reducing the incubation time of the tryptic soy broths before inoculating the Oxoid™ Brilliance MRSA 2 media.
Indirect immunofluorescence assay (IFA) of patient serum on rodent tissue is a European standard for detection of autoimmune liver diseases which comprise autoimmune hepatitis (AIH), primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) as well as their overlapping syndromes. Early diagnosis using anti-smooth muscle (ASMA), anti-mitochondrial (AMA), anti-liver kidney microsome (LKM) antibodies as disease markers allows therapeutic intervention to prevent progression of these diseases. Stomach anti-parietal cell antibodies (APCA) in autoimmune gastritis can also be visualised on rodent tissue.
The aim of this study is to compare three rodent tissue kits: a mouse tissue kit from Menarini and a mouse and rat tissue kit from Werfen, to introduce this technique in the Sint-Jan Bruges hospital. For visualization of the autoantibodies, the classical fluoresence microscopy is compared with the automated fluorescence microscopy, except for the mouse tissue kit from Werfen which was analysed by classical microscopy.
During eight weeks, 50 samples were tested on the rodent tissue kits and analysed by different laboratory workers. The results were compared with those of the UZ Leuven hospital using the mouse Werfen substrate with classical fluorescence microscopy as reference method. Six samples with heterophilic antibodies were tested as well, since these samples are known to produce false positive results on the rodent substrate.
For ASMA, AMA, APCA and LKM antibodies 46 samples corresponded with the reference method using the mouse Werfen substrate and 41 samples with the mouse Menarini substrate, compared to 34 using the rat Werfen substrate. On mouse Menarini slides, three of the six heterophilic antibody samples scored negative, one was falsely classified as ASMA and in three of them no consensus was obtained. While on the Werfen mouse substrate, four samples were falsely classified as ASMA, one was negative and one without consensus. Five samples resulted in aspecific staining and one was without consensus on rat Werfen slides. The results from the mouse tissue from Menarini are about equal between manual and digital fluorescence microscopy. The results of the rat tissue from Werfen were much better with manual analysis than with digital fluorescence microscopy, reason being that there often was not enough rat tissue in the image of the screen for determination.
In conclusion, the results of ASMA, AMA, APCA and LKM antibodies on mouse tissue corresponded better with the mouse reference method than the results obtained on rat tissue. The mouse tissue from Menarini delivered the best result for heterophilic antibodies samples. Future investigations may focus on the comparison of the mouse substrate on the automatic microscopes of both companies.
Cervical Cancer is one of the most common cancers in Belgium with 1414 new cases detected in 2011. The cancer is associated with the human papillomavirus (HPV) which is sexually transmitted. The traditional detection of cervical cancer is only based on the morphological image of the cells, but if the virus is latently present in the cells then is a morphological image unable to detect the virus. The advantages are an early detection of an HPV infection to start an early treatment, a better follow-up for the patient.
The aim of this thesis is to show the importance of co-testing. Co-testing is making a smear and colored with a Papanicoloau staining next to an HPV-testing on a sample with the Cobas 4800 from Roche independently of the result of the Papanicoloau staining.
If the smear is negative, the woman will be adviced to making every three years a new smear. Co-testing will also test for the HPV-DNA and when the sample contains HPV-DNA, the woman will be adviced to come back after one year for follow-up.
In this first part of the study is a global image about human papillomavirus, how works the traditional screening and what is co-testing.
In this second part of the study is tested the precision, reproducibility and a comparison of the positive NILM-samples between the Cobas 4800 and the validated MTA method.
The results in the study showed that 9,7% from the negative samples in the screening has a HPV-infection. There are 468 samples tested independent of the results of the screening (NILM, LSIL and HSIL). There is a 100% sensitivity and specificity with samples of the external quality control. Also, are there 93% of the HSIL samples positive for HPV tested with the Cobas 4800.
The Cobas 4800 is a good method for co-testing. When the comparison is tested between the Cobas and the MTA is the Cobas more sensitive for detect lower concentrations of HPV-DNA. The correctness is also good and is tested with external quality controls and with HSIL-samples.
Myelodysplastic syndrome (MDS) are a group of clonal haematopoietic stem cell diseases characterized by peripheral cytopenia(s) and dysplastic morphology. The diagnosis relies on bone marrow (BM) cytomorphology and cytogenetics. However, recognition of dysplasia in BM smears can be challenging and cytogenetic abnormalities are not always found. On the other hand, the dysplastic features and cytogenetic abnormalities can also be found in conditions other than MDS and in healthy persons. So the diagnosis of MDS is not always straightforward. This thesis aims to evaluate the value of flow cytometry in the diagnosis of MDS.
First there was an evaluation of a flow cytometric score, the Ogata score. The four parameters used in this score are: myeloblasts(%), B-cell progenitors (%), myeloblast CD45 expression, and side scatter of granulocytes. These parameters can be determined with the Lymphoid Screening Tube (LST) that is analysed in every bone marrow sample of a patient without a history of a haematological disorder in AZ Sint-Jan. So, the Ogata score can be determined without an extra cost.
The second method was an extended Ogata score with CD56 expression on the myeloblasts and the monocytes (%). This parameters can also be determined with the LST.
For these methods a total of 37 MDS samples (22 low risk MDS and 15 high risk MDS) and 76 control patients with cytopenia were retrospectively analyzed.
For the third method a new MDS-panel was designed based on literature, in this panel the following parameters were used: CD45, CD15, CD56, CD34, CD11b, CD10, CD19 and CD7 each with a different function. For this prospective evaluation four MDS samples and four control samples were used.
The sensitivity of the Ogata score was 65% and the specificity 92%. For the extended Ogata score a sensitivity of 81% and specificity of 54% was found but their was a problem with an aspecific connection between CD56 and kappa on fluorochroom PE, result in false positive results. With the new MDS panel there wasn’t an added value with the Ogata score to distinguish MDS cases from control cases.
Flow cytometry can be used based on the standard Ogata score as a diagnostic tool for MDS. Because of the high specificity, the diagnosis can be suggested when a positive score is found. Moreover, the analysis can be performed without an extra cost.
Abstract bachelorproef 2 2016-2017: Validation Optilite® CH50 Reagent on Optilite with reference method Wako® Autokit CH50 on P-Modular
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Abstract bachelorproef 3 2016-2017: Is the Idylla device of Biocartis effective for routine use in detecting EGFR / KRAS / BRAF mutations?
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Abstract bachelorproef 1 2015-2016: Evaluation and comparison of four different analyzers for the quantification of HbA1c
To evaluate and compare four commercially available HbA1c analyzers (Menarini HA-8180, Bio-Rad D-100, Tosoh G8 and Sebia Capillarys 2 flex piercing) with respect to (1) correlation against the current laboratory HbA1c analyzer (Menarini HA-8160) and (2) imprecision against the HbA1c analytical goal of the coefficient of variation ≤ 2.9%.
Glycated hemoglobin (HbA1c) has become the preferred method for monitoring diabetes over the last decade. Various commercially available analyzers have been developed for measuring HbA1c in a routine laboratory. The analyzers should deliver fast and accurate results providing both the practitioner and the patient with the information for an appropriate diagnosis, treatment and follow-up of the patient.
The validation and comparison of the four analyzers are performed over a period of 2 weeks. During this period a series of standardized tests are executed and operator observations are recorded. The accuracy of each analyzer, including the laboratory analyzer, is assessed by using the calibrated SKML controls for both a low and a high HbA1c sample. The maximum allowed mean bias is 2 mmol/mol. The repeatability is performed by the consecutive measurement of ten low and ten high samples on each analyzer, whereas the reproducibility is executed by measuring ten identical both low and high samples twice a day during five consecutive days. For both criteria the calculated coefficient of variation CV must be maximum 2.9 %. The linearity is obtained by measuring test samples with linear increasing concentrations of 30 to 80 mmol/mol. A minimum R² of 0.95 is considered to pass this test. In order to verify the correlation of the new analyzers compared to the current laboratory analyzer (Menarini HA-8160) a series of 128 samples, including five hemoglobinopathy samples, is measured. Calculations and graphs of the linear regression with correlation coefficient r, the Intercept A, slope B at a confidence level of 95%, are performed using Passing Bablok regression and Bland Altman plot. Carry-over tests to check how efficiently the analyzer can manage eventual contamination by a previous high HbA1c sample on the next normal samples are executed. Lastly the functionality and the observations made while testing, in particular the features, the complexity, the required preparations, the quality of the reports, the software and the user interface are evaluated.
The coefficient of variation is acceptable for all four analyzers, although the CVs of the repeatability and the reproducibility for both the Menarini HA-8180 and the Tosoh G8 analyzers are significantly lower than for the two other instruments. In respect to linearity only Tosoh G8 (R² = 0.947) is slightly below the target. The Capillarys of Sebia shows the best linearity (R² = 0.9988). Passing-Bablok regression executed on the four analyzers compared with the current Menarini HA-8160 shows only for Sebia an intercept A outside the confidence interval of 95%. Carry-over does not appear on either analyzer.
The analyzer with the best user interface, the least complexity and generating the fastest and the best reports is the Bio-Rad D-100 analyzer.
The Menarini HA-8180 is the best choice considering all criteria with acceptable precision, good linearity and acceptable agreement with the central laboratory results. The more only this instrument provides the feature of obtaining the HbA2 concentration simultaneously with the HbA1c measurement. Also in regard to simplicity of operation, fast and reliable reporting, the Menarini HA-8180 scores high.
In the AZ Sint-Jan Hospital Campus Brugge an HPLC system (Menarini HA-8160) is used for analysis of different types of hemoglobin: HbA1c, HbA0, HbA2, HbF and hemoglobin variants. With this system different clinical manifestations can be detected such as monitoring of patients with diabetes by the quantification of HbA1c and detection of hemoglobinopathies (thalassemias and hemoglobin variants) by quantification of HbA2, HbF and any hemoglobin variants. After thirteen years of duty, this system needs to be replaced, therefore an evaluation of three new HPLC systems and one capillary electrophoresis is performed on demo systems to decide what system would replace the currently used one. In this thesis the evaluation of the four systems for analysis of hemoglobinopathies is displayed. The three HPLC systems are Menarini HA8180T, TOSOH G8 and Biorad D-100. The capillary electrophoresis system is Sebia Capillarys. These where tested on quality control, between run, intra run, correlation, linearity, carry-over and advantages and pitfalls for using them. During these tests 64 blood samples where used, these contained samples with α-and β-thalassemia’s, and hemoglobin variants such as HbA/S, HbS/S, HbA/E, HbA/D-punjab, HbA/C, HbA/Lepore, HbA/J-baltimore and HbA/Muravera. The results showed that every system had its pitfalls and its advantages, although there were no major deviations with clinical significance. This means that in the end every system has successfully passed the evaluation.
Worldwide there were more than 500.000 new diagnoses of cervical cancer in 2012. With a mortality of about 50%, 250.000 women die of this disease annually. Over 99% of these cancers are caused by high risk human papilloma viruses. The quicker the treatment, the better the prognosis. Therefore, the early detection of the virus is very important.
The main purpose of this study is to compare the Cepheid Xpert HPV-assay and the Hologic Cervista HPV-HR test. Both methods test for high risk HPV on cervical PAP-smears. The comparison include hands-on time, runtime, verification and workload.
The Cepheid company has provided 30 HPV high risk screening tests. From our recent files 30 PAP-samples were selected upon which Cervista HPV high risk tests were already performed. The 30 samples include five ASC-US, five NILM, five LSIL and five HSIL-samples. The reproducibility will be tested by testing a proven HPV-positive sample three times on the Cervista MTA and six times on the GeneXpert. The different modules of the GeneXpert will be tested separately.
The HPV-typing of both methods have about the same result. The MTA has a higher sensitivity, sometimes generating false positive results. Because the GeneXpert is single-sample based, the results are immediately available, but generate a higher cost per test. The choice between the two methods depend on the amount of samples that need to be tested. The more samples, the better the MTA is for a technique. In case of only a few samples a day the GeneXpert is preferred, but with a higher cost.
- Cut- off value
- Dilution of the DNA extract to a DNA concentration of 2ng/µl.
- ‘Polymerase chain reaction’ step: specific DNA sequences are amplified to generate enough DNA, necessary for analysis.
- Pyrosequencing with the PyroMark Q24: with this method, the sequence of nucleotides in DNA can be defined. Mutations can be detected in the RAS-genes.
Dr. Ivo Van Den Berghe
Dr. Jacques Van Huysse
Dr. Jan Emmerechts
Dr. Michel Langlois