The continuous improvement of the quality of the tests used in a medical context is an important aspect of modern, clinical laboratories. One of the ways of achieving this objective is by continuously (re)evaluating and improving the reagents used in the lab. This study therefore aims to compare and validate different reagents for various thrombophilia tests currently performed in the laboratory of AZ groeninge. The main objective of this study is to obtain an effective and reliable test for the detection of protein C, protein S and antithrombin deficiency and for the screening of haemostasis anomalies like activated protein C resistance (APCr). These tests are helpful for an accurate and effective treatment of thrombophilia patients and therefore they should be examined and tested in detail. Multiple firms, namely Siemens, Hyphen Biomed and Werfen offer reagents for the measurement of these four parameters in citrate plasma samples.
The validation of each reagent is achieved by determining the repeatability, reproducibility, bias and total error of each test. The various tests are also checked for the effect of possible interfering components like a highly haemolytic sample or the presence of the anaesthetic propofol in the citrate plasma. A method comparison is also performed between the different reagents. Finally a comparison is made of both the cost and shelf life of each reagent. All measurements are performed on the Sysmex CS2100i. After gathering the above mentioned data, the most optimal test will be recommended for usage in the laboratory of AZ groeninge.
The following results have been obtained:
The Werfen COAMATIC Protein C assay is the most recommended assay for the measurement of protein C levels in plasma. Both the total error and the precision have been determined to be the best of the three examined assays. In addition of this great performance, the shelf life of this test (three months at 2-8°C) is longer than the shelf life of any other examined reagent.
The INNOVANCE Free PS Ag assay of Siemens is the best choice for the evaluation of protein S. It shows great performance on the CS2100i and has other good characteristics like low cost and a decent shelf life.
For the detection of antithrombin deficiencies, the tests offered by Hyphen Biomed and Siemens are nearly equal in both performance and cost/shelf life.
Because of a continual calibration failure due to an unknown cause, the APCr assay of Hyphen has not been taken into account for the final choice of APCr test. The Siemens ProC Global test shows less false positive results and is less influenced by the presence of propofol in a patient’s blood. However, the performance of the ProC Ac R test is better than the ProC Global test. Therefore the laboratory itself should decide which of the two ProC assays has the most significant advantage when used in the lab.
Due to the limited availability of reagents and patient samples, the method comparison has only been performed on a low amount of samples. If possible the laboratory method comparison should be reperformed in the future on a higher amount of citrate plasma samples. It is also recommended to evaluate the reliability of the reference values and the cut off values mentioned in the leaflets of each company before applying them in the laboratory.
The aim of this research is to validate a new Autostainer Link 48 from the firm Agilent as replacement for the old platform. The aim of this research is to secure the correct sample staining before analysing analyse patient samples. The Autostainer Link 48 stains paraffin slides using immunohistochemistry. For this study, four parameters being tested: repeatability, reproducibility, correctness, homogeneity.
To test the repeatability, antibodies such as CD7, CK7, CD45, CK PAN, Ki67 being stained all together in one run, on three several days. There is one slide of every antibody in the run. That’s different with the reproducibility, there are three slides of every antibody being stained in one run. The antibodies being stained for reproducibility are the same as the ones stained for repeatability. Reproducibility is performed on one day, not on several days.
The correctness is examined by staining with antibodies such as PMS2, MLH1, MSH2, MSH6, CD117, S100, PDL-1, P53, BCL6 one time in a run. Last but not least there is the homogeneity as an examined parameter. Therefor, an antibody such as CK PAN or vimentin is placed in the staining platform on all 48 sites, to check if there is a homogeneous staining.
As a result of the study the four parameters were approved by the pathologists. At the end of the study, because all parameters were approved, the Autostainer was released for analysing patient samples. The validation process was carried out before the old Autostainer was removed
Validation of procalcitonin analyses. The three kits (Vidas Brahms PCT, Elecsys Brahms PCT, and PCT LiquiColor Assay) were evaluated and compared to each other to choose the best method for routine use.
There is more and more interest in procalcitonin as a biomarker for sepsis due to its advantages against others conventional markers. Procalcitonin is a good marker for bacterial infection such as sepsis and that does not increase in case of viral infection. Furthermore, procalcitonin provides a fast diagnosis which is very important for sepsis patients.
The kits were compared on the basis of the repeatability, reproducibility and the trueness. Three concentration levels (negative, weakly positive and strongly positive) were measured ten times on the same day for the intra-run imprecision. For the inter-run imprecision, the three concentration levels were measured one time a day on ten different days. The intra-run and the inter-run of the methods have to meet the acceptation criterium of standard deviation <0,0375 ng/ml procalcitonin. Samples of sepsis patients, patients infected by influenza/RSV, patients on meropenem and intensive care patients were used for the trueness. The trueness of the methods was evaluated based on the absolute bias plot with an acceptation criterium of ± 0,075 ng/ml and on the passing-bablok with confidence intervals of 95%.
The repeatability and the reproducibility of Vidas Brahms PCT met the acceptation criterium for the three concentration levels. Only the negative and weakly positive concentration levels of Elecsys Brahms PCT met the criterium. For the PCT LiquiColor Assay, the weakly positive and strongly positive did not meet the criterium of the repeatability and the repeatability of the negative concentration level can not be measured because the results were below the lower limit of quantification. Therefore, no further research was being done for PCT LiquiColor. Vidas Brahms PCT and Elecsys Brahms PCT both did not meet the criterium for the trueness.
According to the results of the repeatability and reproducibility, it can be concluded that the Vidas Brahms PCT is better than the other two methods and suitable for use in the routine laboratory. However, the trueness of this method did not meet the criterium. The main reason for choosing the Vidas Brahms PCT is its lower price.
Abstract bachelorproef 2 2016-2017: Comparative study of the EGFR test on the IdyllaTM
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Tryptase is an enzyme, made and stored in mast cells. The measurement of tryptase is a useful marker in the diagnosis of systemic mastocytosis, allergic reactions and anaphylaxis.
Tryptase can be measured with a Phadia system. These days AZ Groeninge sends the samples for measuring tryptase to UZ Leuven. But AZ Groeninge would like to investigate the possibility of determining tryptase in their own laboratory. So the purpose is to validate the tryptase assay on the Phadia 250 system in the laboratory of AZ Groeninge.
For the validation of the Phadia 250 system of AZ Groeninge, a few performance characteristics were evaluated: precision (reproducibility and repeatability), trueness, uncertainty of measurement, method comparison and measurement range. Also the stability of tryptase in serum was evaluated.
For the evaluation of the reproducibility and the trueness the tryptase level of the Tryptase Control and the Curve Control was measured on ten different days. The coefficient of variation (CV) and the relative bias (Brel) were calculated. The CV is used to evaluate the between-day reproducibility and the Brel to evaluate the trueness. The CV was lower than the proposed 10% and the Brel was situated between -10% and +10%.
Two patient samples, one with a high tryptase level and one with a low tryptase level, were used for the evaluation of the repeatability. The concentration of tryptase was measured ten times in the same run. The within-run precision was acceptable with a CV lower than 10%.
The uncertainty of measurement (Total Error (TE) = Brel + 1,65 * CV) was determined with the previous results. The TE of both controls was lower than the calculated error limit of 26,5%.
Since there was no reference method available, we compared results of 32 samples that were determined in UZ Leuven on a Phadia 1000 system. A Wilcoxon test showed a statistically significant bias. Passing-Bablok regression analysis showed a significant slope and a non-significant intercept. However, the difference was clinically acceptable according to the absolute difference plot between the two methods.
The manual of the Phadia 250 system states that the machine can measure tryptase levels between 1 and 200 µg/L. Calibrators were used as samples and the Brel was calculated. With a Brel between ±15% the measuring range between 1 and 200µg/L was confirmed.
Regarding stability tryptase should be measured within five days if a sample is stored in the refrigerator according to the manufacturer. Tryptase was measured on five new samples and then stored in the refrigerator. Tryptase was measured again after five and seven days. There was no statistically significant difference between the results from day one, five and seven. Thus samples can be stored for up to seven days in the refrigerator.
The conclusion is that the Phadia 250 system in AZ Groeninge can be used for the routine measurement of tryptase in serum. It showed acceptable performance characteristics regarding precision, trueness, measurement uncertainty, method comparison, measuring range and stability.
Abstract bachelorproef 2 2015-2016: Development of controls for immunohistochemical staining
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Dr. Alain Vanneste
Annelies De Bel