User manual
Contents
1. TUBE T DESCRIPTION LABEL OR LID COLOUR ee RT MIX 3x143uL 6x135uL 6x260 pL Reverse transcriptase RT Enzyme 1x17 uL 1x 30 uL 2 x 30 uL Mastermix 2X 2X Q Real time mix 2x350uL 3X450uL 6X 450 uL Magnesium Chloride solution MgCl 1x50 uL 1X75 ul 1X 150 uL Primer and probe Mix WT 1 Oligomix White 1x27 uL 2x27 uL 4 x 27 uL Primer and probe Mix ABL Oligomix Blue 1x27 uL 2x27 uL 4 x 27 uL RQ S57 48 EN doc 4 Quen 3 STORAGE AND STABILITY OF THE REAGENTS Each component of the kit must be stored according to the directions indicated on the label of of each box In particular Box P Store at 30 C 20 C If stored at the recommended temperature all test reagents are stable until their expiration date The 2X Q Real time Mix and Oligomix are sensitive to physical state variations it is recommended not to let the reagents undergo more than two freeze thaw cycles If the single test runs are limited to a small number of samples it is recommended to aliquot the reagents 2X Q Real time Mix and Oligomix contain fluorescent molecules it is recommended to store these reagents away from direct light 4 PRECAUTIONS FOR USE The kit must be used only as an IVD and handled by qualified technicians who are well educated and trained in molecular biology techniques applied to diagnostics Before starting the kit procedure read carefully and completely the user manual Keep2. for the WT 1 were tested simultaneously with the REALQUALITY RS WT 1 kit and another CE IVD or reference method From the obtained results the diagnostic sensitivity of this device was calculated to be 97 15 7 Accuracy This value was calculated by the number of correct amplifications over the total number of executed amplifications The REALQUALITY RS WT 1 device has an accuracy of 98 4 Qamauitica 25 RQ S57 48_EN doc 16 REFERENCES Cilloni D et al Haematologica 93 6 921 924 2008 Freeman SD et al Semin Oncol 35 4 388 400 2008 Gabert J Beillard E et al Leukemia 17 12 2318 57 2003 Liu Yin JA Best Pract Res Clin Haematol 15 1 119 35 2002 Saiki RK S Scharf F Faloona KB Mullis GT Horn HA Erlich and N Arnheim Science 230 1350 1354 1985 Sugiyama H Int J Hematol 73 2 177 87 2001 Van der Velden VH et al Leukemia 17 1013 1034 2003 van Dongen JJ et al Lancet 352 1731 1738 1998 van Dongen JJ Macintyre EA Gabert JA et al Leukemia12 1901 1928 1999 Verfaillie CM Biology and therapy of chronic myelogenous leukaemia vol 12 num 1 1998 17 USEFUL LINKS www hematology org www bloodjournal org www bloodline net www haematologica it www il st acad sci org data6 html http medocs ucdavis edu IMD 420A dib index htm http web tiscali it ematologia www ematologia italia net frame_b htm www simti it http stemcells alphamedpress org www blacksci co uk uk society bsh RQ S57 48 EN doc 26
3. uL of cDNA or 5 uL of positive control provided in the kit RQ S57 48 EN doc 16 Qanaurtica www abanalitica it Always amplify a negative control together with the samples to be analyzed add sterile water instead of extracted DNA to the corresponding well both for the WT 1 and the ABL Mix Hermetically seal the plate by using an optical adhesive film or appropriate sealer Make sure that there are no air bubbles in the bottom of the wells and or centrifuge the plate at 4000 rpm for about 1 minute Load the plate on the instrument making sure to position it correctly and start the amplification cycle 11 5 ANALYSIS OF THE DATA AND INTERPRETATION OF RESULTS At the end of the reaction view the graph in logarithmic scale Figure 2 Analyze WT 1 and ABL graphs and quantification results separately Position the Threshold by choosing the position in which the Correlation Coefficient R and the slope of the curve values are the closest possible to 1 and 3 33 respectively Figure 3 Results are considered acceptable when the efficiency of the amplification is between 85 110 slope approximately 3 75 3 10 and the Correlation Coefficient value is not less than 0 990 Qanautica 17 RQ S57 48_EN doc Deta An vs Cycle Fig 2 Analysis of the post run data amplification graph of WT 1 in lo
4. 5 25 25 26 26 27 anauitica Case iItica it www abanaliti 1 PRODUCT INFORMATION 1 1 Intended Use The REALQUALITY RS WT 1 is an IVD for identification and quantification of the Wilm s Tumor gene WT 1 expression located on chromosome 11p13 by amplification of cDNA in the regions of the gene on exon 1 2 If used together with the REALQUALITY RQ WT 1 STANDARD code RQ 58 ST kit it allows the quantification of the WT 1 transcripts present in the sample The Real time PCR amplification method is used in this kit starting from c DNA obtained by reverse transcription of the RNA extracted from human clinical samples This in vitro diagnostic test is an auxiliary device for detection monitoring and reoccurrence of hematopoietic leukemia cells above all in cases with Acute Myeloid Leukemia AML It is recommended to use this kit as indicated in the instructions herein The present manual refers to the following product REALQUALITY RS WT 1 Kit for identification and quantification of the Wilm s Tumor gene WT 1 expression by Real time PCR amplification This product is in accordance with 98 79 CE Directive Annex Ill regarding the n Vitro medical diagnostic devices CE mark Contains all the reagents needed for Real time amplification Code Product PKG RQ S57 48 REALQUALITY RS WT 1 48 test RQ S57 96 REALQUALITY RS WT 1 96 test Qamautica 2 RQ S57 48_EN doc 2 KIT CONTENT STORE AT 30 20 C
5. ABLcn copy number NCNwr 1 WT 1cy ABLen x 10 The Minimal Residual Disease MRD is expressed as the ratio between the WT 1 normalized copy number at the follow up FUP and the WT 1 normalized copy number at the time of the diagnosis DX MRD NCNeup NCNpx In case of the follow up samples the sensitivity SENSv of the experiment must be calculated in order to determine the clinical validity of the obtained results SENSv ABLen px ABLen tup X WT 1cn px RQ S57 48 EN doc 20 Qanauitica 13 TROUBLESHOOTING Absence of amplification signal for positive controls standards and samples e The instrument was not programmed correctly Repeat the amplification paying attention of the instrument programming pay particular attention to the thermal profile the selected fluorophores and the correspondence between the plate protocol and the plate itself e The amplification mix was not prepared correctly Prepare a new amplification mix paying attention to follow the instructions reported in paragraph 11 4 e The kit was not stored properly or it was used past the expiration date Check both the storage conditions and the expiration date reported on the label use a new kit if needed Weak amplification signal intensity for positive controls standards e Positive controls standards were stored incorrectly and have degraded Store the positive controls standards correctly at 2 C 8 C Do not use the positive
6. ANALITICA ADVANCED BIOMEDICINE User manual REALQUALITY RS WT 1 code RQ S57 Kit for identification and quantification of the Wilm s Tumor Gene WT 1 expression CE RQ S57 48 EN doc 1 PRODUCT INFORMATION 3 1 1 Intended Use 3 2 KIT CONTENT 4 3 STORAGE AND STABILITY OF THE REAGENTS 5 4 PRECAUTIONS FOR USE 5 5 SAFETY RULES 7 5 1 General safety rules 5 2 Safety rules about the kit 7 6 MATERIALS REQUIRED BUT NOT PROVIDED 8 6 1 Reagents 8 6 2 Instruments 8 6 3 Materials 8 7 INTRODUCTION 9 8 TEST PRINCIPLE 10 9 PRODUCT DESCRIPTION 12 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES 13 11 PROTOCOL 13 11 1 RNA extraction 13 11 2 Reverse transcription RT for the cDNA synthesis 14 11 3 INSTRUMENT PROGRAMMING 15 11 3 1 Creation of thermal protocol 15 11 3 2 Plate Setup 15 11 4 AMPLIFICATION PROTOCOL 16 11 5 ANALYSIS OF THE DATA AND INTERPRETATION OF RESULTS 17 12 NORMALIZATION AND QUANTIFICATION OF MINIMAL RESIDUAL DISEASE20 Qanaurtica 1 RQ S57 48_EN doc www abanaliti 13 14 15 15 1 15 2 15 3 15 4 15 5 15 6 15 7 16 17 18 TROUBLESHOOTING DEVICE LIMITATIONS DEVICE PERFORMANCES Analytical specificity Analytical sensitivity detection limit Analytical sensitivity linearity Reproducibility Diagnostic specificity Diagnostic sensitivity Accuracy REFERENCES USEFUL LINKS RELATED PRODUCTS RQ S57 48_EN doc 2 21 23 23 23 23 23 24 2
7. Qanauitica 18 RELATED PRODUCTS REALQUALITY RQ WT 1 STANDARD Quantified and ready to use quantification standard for WT 1 Code Product PKG REALQUALITY 4 x 60 uL WT 1 RQ 58 ST RQ WT 1 STANDARD 4 x 60 uL ABL Qanaurtica 27 RQ S57 48_EN doc RQ S57 48 EN doc 28 ANALITICA www abanalitica it ANALITICA ADVANCED BIOMEDICINE www abanalitica it AB ANALITICA srl Via Svizzera 16 35127 PADOVA ITALY Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it
8. amplification reaction is still proportional to the number of target molecules in the solution The starting concentration of the unknown samples is determined by comparison of the Ct value of each sample with the Ct value of a standard curve acquired at known concentration Figure 1 RQ S57 48 EN doc 10 Qanauitica RA LZ NN 200 NE 0 2 4 6 ENG 28 30 32 34 36 36 40 42 44 46 Cycle Correlation Coefficient 0 999 Slope 3438 Intercept 28 768 Y 3 338 X 38 68 n Unknowns PCR Efficiency 99 3 o Standards Threshold Cycle a RO N RO IO CO C2 CO D HO IO RO SN Log Starting Quantity copy number Figure 1 Creation of a standard curve starting from the standard Ct values at known concentration Main advantages of the Real time PCR technique compared to the conventional amplification techniques are for example the possibility to execute a semi automated analysis in which the time needed for the visualization of the amplicons is eliminated and the absence of the post amplification sample manipulation that reduces the possible contamination phenomena Qanautica 11 RQ S57 48_EN doc www abanalitica it 9 PRODUCT DESCRIPTION The REALQUALITY RS WT 1 code RQ S57 used with the REALQUALITY RS WT 1 STANDARD code RQ S58 ST allows the identification and quantification of Wilm s Tumor WT 1 gene expression localized on chromosome 11p13 by c DNA amplification of the gene regions on exons 1 2 Such quantific
9. ation is obtained by the construction of a four point standard curve for WT 1 and in parallel for ABL genes In fact starting from the c DNA itself but in a separated PCR reaction the sequence of housekeeping gene ABL is amplified such amplification in addition to be a mark for the quantification and normalization allows to evaluate both the extracted RNA suitability the following retro transcription reaction and the possible presence of PCR reaction inhibitors This valid tool helps the user to recognize possible false negative results ABL gene amplification is made separately from WT 1 amplification because experimental evidences demonstrate that a competition between the two targets can occur in samples with a low number of WT 1 transcripts and sometimes it ends up to heavily disadvantage the specific translocation transcript amplification with the possibility to have false negative The given positive controls are made by a DNA fragment with the target region of interest and they are not dangerous for the user For amplification reaction preparation a ready to use Mastermix is supplied containing all the reagents needed with the exception of the Oligomix and in particular e ROX an inert colorant in which the fluorescence does not undergo changes during the amplification reaction it is used to normalize eventual differences between wells caused by artifacts from pipetting errors or instrument limitations e dUTP UNG system pr
10. ch position ROX is an inert colorant in which the fluorescence does not undergo changes during the amplification reaction on instruments that use ROX Applied Biosystems Stratagene etc it is used to normalize eventual differences between wells caused by artifacts due to pipetting errors or instrument limitations Record where required that the final reaction volume is 25 uL Qamauitica 15 RQ S57 48_EN doc 11 4 AMPLIFICATION PROTOCOL Once thawed mix the reagents by inverting the tubes several times do not vortex then centrifuge briefly Prepare the reaction mix rapidly at room temperature or work on ice or on a cooling block Try when possible to work in area away from direct light Prepare as described below a mix sufficient for all the samples to be tested counting also for the positive and negative control in the latter H20 is added instead of DNA and when calculating the volume consider an excess of at least one reaction volume WT 1 Amplification Reagent 1 Reaction 2X Q Real time Mix 12 5 uL WT 1 Oligomix 1 0 uL MgCl 0 5 uL H20 6 0 uL Total Volume 20 0 uL Reagent 1 Reaction 2X Q Real time Mix 12 5 uL ABL Oligomix 1 0 uL MgCl 0 5 uL H20 6 0 uL Total Volume 20 0 uL Mix by inverting the tubes in which the mix was prepared in several times Then centrifuge briefly Pipette 20 uL of the mix in each well of the plate Add to each well in the correct positions 5
11. controls standards past the expiration date e The reaction mix does not function correctly Make sure to store the 2X Q Real time Mix and Oligomix correctly at 20 C 30 C Avoid unnecessary freeze thaw cycles Amplification signal of ABL very delayed or absent in the extracted samples e The extracted RNA is not suitable for amplification or a problem may have occurred during the reverse transcription reaction and the amplification reaction was inhibited Make sure to perform the extraction of nucleic acids correctly Qamaurtica 21 RQ S57 48_EN doc If an extraction method uses wash steps with solutions containing Ethanol make sure no Ethanol residue remains in the DNA sample Use the extraction methods suggested in paragraph 11 1 During reverse transcription reaction check that the reverse transcriptase enzyme has been pipette in the tube by looking for the drop formed by the enzyme on the tube wall after being added to the mix then centrifuge briefly Follow standard procedures for minimizing RNA degradation use RNase free plastic lab ware and work on ice during the reverse transcription reaction For any further problems please contact AB ANALITICA s technical support at laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 RQ 57 48_EN doc 22 auos 14 DEVICE LIMITATIONS The kit can have reduced performances if e the clinical sample is not suitable for this analys
12. dy leaded to a better comprehension of the cellular and molecular mechanisms involved in many neoplastic pathologies thanks to the easy finding and sampling of malignant leukemic cells present in the blood despite the solid tumor s ones which are sometimes difficult to be collected without the use of invasive methods The WT 1 gene is localized on chromosome 11p13 encode for a transcription factor containing four zinc finger domains in the C terminal part and it was initially identified for its role in the pathogenesis of Wilm s tumor Sugiyama et al 2001 Moreover it has been demonstrated that WT 1 gene is highly expressed in most of hematopoietic tumor pathologies Sugiyama et al 2001 Liu Yin et al 2002 in samples of peripheral or bone marrow blood of healthy subjects indeed WT 1 shows low expression levels such levels are increased instead in a significant way in patients with acute myeloid leukemia AML and acute lymphoblastic leukemia ALL both in pediatric and adult age even in subjects with chronic leukemia The scientific community thought to establish a cut off for the sample definition at high subjects with disease and low healthy people expression of WT 1 Cilloni D et al Haematologica 2008 Mean Value WT1 Range copies per dl ABL copies Normal bene marrow eM 5 o Normal peripheral blood PB 5 020 Different studies highlighted the importance of the monitoring of the Minimal Residual Di
13. eagents for density gradient separation of mononucleated cells Ficoll RNA extraction reagents Dnase and Rnase free sterile water Distilled water REALQUALITY RQ WT 1 STANDARD code RQ 58 ST for quantitative analysis 6 2 Instruments e Laminar flow cabinet its use is recommended while preparing the amplification mix to avoid contamination it would be recommended to use another similar laminar flow cabinet to add the extracted DNA and standard solutions e Micropipettes range 0 5 10 uL 2 20 pL 10 100 uL 20 200 pL 100 1000 uL e Microcentrifuge max 12 14 000 rpm e Plate centrifuge optional e Thermalcycler for reverse transcription e Real time amplification instrument The kit was standardized on Applied Biosystems 7500 Fast Dx 7300 StepOnePlus Real Time PCR System Applied Biosystems the kit can be utilized on instruments that use 25 uL of reaction volume and can detect the FAM fluorescence correctly For further information on instrument compatibility of the kit please contact AB ANALITICA s technical support 6 3 Materials e Talc free disposable gloves e Disposable sterile filter tips range 0 5 10 uL 2 20 pL 10 100 uL 20 200 uL 100 1000 uL e Sterile DNase and RNase free tubes for reverse transcription e 96 well plates for Real time PCR and the optical adhesive film or 0 1 0 2 mL tubes with optical caps RQ S57 48 EN doc g Qanauitica 7 INTRODUCTION The leukemia and lymphoma stu
14. events contaminations from previous amplifications since it removes residual uracil incorporated in the molecule of single or double stranded DNA NOTE The kit was developed in accordance with the Europe Against Cancer guidelines Gabert et al Leukemia 2003 and with the international recent recommendations Branford et al Leukemia 2006 RQ S57 48 EN doc 12 anaurica 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES The analysis of the WT 1 expression is performed starting from whole peripheral or bone marrow blood Sample collection must follow all the usual sterility precautions Blood must be treated with EDTA Other anticoagulation agents as heparin are strong inhibitors of TAQ polymerase and so they could alter the efficiency of the amplification reaction Fresh blood can be stored at 2 C 8 C if processed in 4 hours after the withdrawal thus it is necessary to proceed with the mononucleate cells separation by density gradient centrifugation Ficoll reagent not included in the kit From the pellet of lymphocytes obtained as such is possible to proceed directly with RNA extraction otherwise the cell pellet may be conserved at 80 C until the RNA extraction better if preserved in a buffer containing RNAse enzyme inhibitors i e RLT buffer QIAGEN or Trizol 11 PROTOCOL 11 1 RNA extraction The product was validated using the RNeasy Mini kit QIAGEN Hilden Germany For use follow the user ma
15. fly and incubate in a thermalcycler programmed as below 20 C 10 min 1 cycle 42 C 45 min 99 C 3 min 4 C 5 min Once the cycle ends add 30 uL of sterile water to each retrotranscribed sample The diluted cDNA can be stored at 2 C 8 C for short period of time maximum one week or at 20 C 30 C for longer periods of time NOTE the cDNA obtained can be used both for the WT 1 translocation amplification and for the housekeeping ABL gene amplification RQ S57 48 EN doc 14 Qanauitica 11 3 INSTRUMENT PROGRAMMING 11 3 1 Set the following thermal profile Creation of thermal protocol Cycle Repeats Step Time UNG Activation 1 1 1 2 00 50 0 Taq Activation 2 1 1 10 00 95 0 Ampincaton 3 45 1 00 15 95 0 cycles 2 01 00 60 0 Fluorescence collection step 11 3 2 Plate Setup Define the dilution of the WT 1 and ABL standard in the interval from 10 to 10 copies Mark the grid of the new plate with the position of the negative control NTC standards STD and samples Unknown making sure the position is the same as on the plate and identify each sample with its name NOTE it is recommended to amplify both samples and positive negative controls and standards in duplicate Select and activate the FAM fluorophore and NONE as quencher Pay attention that for the instruments that require it the detection of the fluorescence of the fluorophore ROX corresponds to ea
16. garithmic scale view on Applied Biosystems 7300 Real Time PCR System with SDS software version 1 2 3 Fig 3 Analysis of the post run data standard curve visualization on Applied Biosystems 7300 Real Time PCR System with SDS software version 1 2 3 RQ S57 48_EN doc 18 Qavaurica ti www abanalitica it NOTE If one works in duplicates it is important that the replicates do not produce a difference of the Ct ACt gt 1 5 For ABL values samples with ABL Ct corresponding to the copy number that is inferior to the minimum limit of the linearity range see paragraph 15 Device performances must be excluded from the analysis The International scientific community also defined an ABL Ct range within which the samples can be considered adequate for the analysis ABL Ct 21 8 29 4 J Gabert et al Leukemia 2003 This is of particular importance when studying the Minimal Residual Disease in samples with a low WT 1 copy number it allows to be sure that the obtained results is correct and to exclude the possibility that a low copy number of WT 1 is due to low cells number in the samples Qamautica 19 RQ S57 48_EN doc 12 NORMALIZATION AND QUANTIFICATION OF MINIMAL RESIDUAL DISEASE The WT 1 and ABL standard curves allow to transform the Ct values obtained for unknown samples in WT 1 WT 1 cn and ABL ABLen copy numbers The normalized copy number NCN of the WT 1 transcript is defined as the ratio between the WT 1 cy and
17. is sampling and or storage error i e heparin treated blood or use of other unsuitable anticoagulants e the treatment of the starting sample was not performed as indicated in the paragraph 10 e the kit was not stored correctly 15 DEVICE PERFORMANCES 15 1 Analytical specificity The specificity of the REALQUALITY RS WT 1 code RQ S57 kit is guaranteed by an accurate and specific selection of primers and probes and also by the use of stringent amplification conditions The alignment of primers and probes in the most important databanks shows the absence of non specific pairing 15 2 Analytical sensitivity detection limit The analytical sensitivity limit of REALQUALITY RS WT 1 kit was defined by the amplification test of 8 dilution replicates from the last point of the quantification standard conducted in at least 3 consecutive runs The results are reported in Table 1 15 3 Analytical sensitivity linearity The linearity of the assay was determined using a quantification standard panel The analysis of the data obtained by linear regression have demonstrated that the test presents for WT 1 and for ABL a linear response for all the panel point R gt 0 99 The results of the analysis are reported in Table 1 Qamauitica 23 RQ S57 48_EN doc 15 4 Reproducibility A 50 transcript copies uL dilution corresponding to a final amount of 250 transcript copies reaction of the quantification standard was amplified in eight replicate
18. nual of the manufacturer However the device is suitable for most diffused manual or automatic RNA extraction methods For any further information on device compatibility with different extraction methods please contact AB ANALITICA s technical support Please follow the instructions below regarding the quantity of RNA to be used for the reverse transcription reaction about 1g Qamauitica 13 RQ S57 48_EN doc 11 2 Reverse transcription RT for the cDNA synthesis Attention before starting the reverse transcription procedures it is recommended to use an ice container and to thaw one or more RT Mix aliquots depending on the number of analyzed samples Once thawed the RT Mix must be mixed well by inverting the tube several times do not vortex and after a brief centrifuge must be stored on ice until use For each sample add to a sterile DNase and Rnase free tube see paragraph 6 3 Extracted RNA 5 uL NOTE The 5 uL amount indicates the void volume available for the reaction The appropriate amount of RNA to be used for reverse transcription is about 1 ug if the RNA is more concentrated it is necessary to dilute it properly with DEPC H O Insert the tubes in the thermalcycler and program the following thermal profile 1 cycle 70 C 10 min Next place the tubes on ice immediately for at least 5 minutes Add 14 5 pL of RT Mix and 0 5 uL of RT Enzyme mix by pipetting centrifuge brie
19. ogy represents an advancement of the basic PCR technique it allows to measure the number of DNA molecules amplified during the exponential amplification phase The amplicon monitoring is essentially based on the labeling of the primers and probes or of the amplicons themselves with fluorescent molecules In the first case the Fluorescence Resonance Energy Transfer FRET among the two fluorophores or other mechanisms which lead to fluorescence emission and involve a fluorophore and a non fluorescent quencher molecular beacon scorpion primer etc are used The mechanism that determines the fluorescence emission is based on the presence of a quencher molecule located in proximity of a reporter molecule that blocks the fluorescence emission by the reporter When the quencher is separated from the reporter the latter emits fluorescence The real time detection of such fluorescence is accomplished by means of a thermalcycler equipped with fluorescence detector Each amplification cycle will release a certain amount of fluorescence into the solution the cycle at which the amplification generates the minimal amount of fluorescence needed to overcome the basal noise threshold is called the cycle threshold Ct By intuition the higher the starting concentration of the target nucleic acid the sooner the amplification will reach the cycle threshold The Ct value is reached during the exponential phase of the amplification reaction where the
20. s in the same run in order to determine the intra assay variability variability among the replicates of a certain sample in the same assay The intra assay variability coefficient of the method in respect to the Cycle threshold Ct for WT 1 and ABL is reported in Table 1 The last point of the quantification standard 20 transcript copies uL corresponding to 100 transcript copies reaction was amplified in duplicates in three consecutive runs in order to determine the inter assay variability variability of the replicates of the same sample in different runs For each run the variability coefficient was calculated from the Ct of the samples The inter assay variability coefficient for WT 1 and ABL was calculated from the average of the variable coefficients in each experiment performed and is reported in Table 1 ABI 7500 StepOne Detection Limit 10 5 copies reaction 100 positive 100 positive Table 1 Linear Range copies reaction Intra assay Variability 0 916 0 669 0 454 inter assay Variability 0 328 0 158 0 334 RQ 57 48_EN doc 24 mue 15 5 Diagnostic specificity A significant number of samples negative for WT 1 were tested simultaneously with the REALQUALITY RS WT 1 kit and another CE IVD or reference method From the obtained results the diagnostic specificity of this device was calculated to be 100 15 6 Diagnostic sensitivity A significant number of samples positive
21. sease MRD by quantitative Real time PCR in order to highlight specific molecular markers of the disease for example the fusion transcripts BCR ABL and AML1 ETO or mutations as NPM 1 this enables for the individuation of patients at high risk relapse for whose the precocious therapeutic intervention is extremely important Freeman et al 2008 Since for an half of the AML patients is not possible to detect a leukemia specific target it is extremely important to develop alternative approaches so that the MRD is applicable to a large number of patients A lot of studies demonstrated the utility of quantitative Real time PCR in order to monitor the expression levels of WT 1 gene as relative indication for the leukemic diagnosis for aggressiveness level of the disorder and to the response to the pharmaceutical treatment Freeman et al 2008 Qamauitica 9 RQ S57 48_EN doc 8 TEST PRINCIPLE PCR method Polymerase Chain Reaction was the first method of DNA amplification described in literature Saiki RK et al 1985 It can be defined as an in vitro amplification reaction of a specific part of DNA target sequence by a thermostable DNA polymerase This technique was shown to be a valid and versatile molecular biology instrument its application contributed to a more efficient study of new genes and their expression and it brought to a revolution in the laboratory diagnostic and forensic medicine field The REAL TIME PCR technol
22. the kit away from heating sources One must pay particular attention to the expiration date on the label of each box do not use any part of the kit past the expiration date The reagents present in the kit must be considered an undividable unit Do not divide or use different reagents from other kits or lots All the reagents must be thawed at room temperature before use It is recommended to do not vortex but to mix the solutions by inverting the tube several times and then centrifuge them briefly Prepare the reaction quickly at room temperature or work on ice or on a cooling block Qamauitica 5 RQ S57 48_EN doc In case of any doubt about the storage conditions box integrity or method application please contact AB ANALITICA s technical support at laboratorio abanalitica it During nucleic acid amplification the technician has to take the following special precautions Use filter tips Store the biological samples the extracted RNA cDNAs and positive controls included in the kit and all the amplification products in different places from where amplification reagents are stored Organize the work areas in different pre and post PCR units do not share instruments and consumables pipettes tips tubes etc between them Change gloves frequently Wash the bench surfaces with 5 Sodium Hypochlorite Keep the RNA just extracted or that will be stored at 30 C 20 C or 80 C according to the time required be
23. tween extraction and reverse transcription on ice during reverse transcription preparation RQ S57 48 EN doc 6 Qanauitica SAFETY RULES General safety rules Wear disposable gloves to handle reagents and clinical samples and wash hands at the end of the procedure Do not pipette by mouth Since no known diagnostic method can assure the absence of any infective agents it is a good rule to consider every clinical sample as potentially infectious and handle it as such All devices that come in contact with clinical samples should be considered as contaminated and disposed of as such In case of accidental spilling of the samples clean up with 10 Sodium Hypochlorite The materials used to clean up should be disposed in special containers for contaminated products Clinical samples materials and contaminated products should be disposed of after decontamination immerse in a solution of 5 Sodium Hypochlorite 1 volume of 5 Sodium Hypochlorite solution for every 10 volumes of contaminated fluid for 30 minutes OR autoclave at 121 C for at least 2 hours NOTE do not autoclave solutions containing Sodium Hypochlorite 5 2 Safety rules about the kit The risks for the use of this kit are related to the single components Dangerous components none The Material Safety Data Sheet MSDS of the device is available upon request Qamaurtica 7 RQ S57 48_EN doc 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 Reagents R
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