A.Z. Sint-Jan Brugge-Oostende AV campus Sint-Jan
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