User manual REALQUALITY RS-AML1
Contents
1. NN mo 23 o 0 200 0 2 4 6 8 10 12 14 16 18 20 22 28 30 32 34 36 38 40 42 44 46 Correlation Coefficient 0 999 Slope 3438 Intercept 45 768 Y 3 338 38 N68 n Unknowns PCR Efficiency 99 3 Standards 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 anauitica ADVANCED BIOMEDICINE www abanalitica it RQ S59A 48 EN doc 12 9 PRODUCT DESCRIPTION The REALQUALITY RS AML1 ETO without RT reagents code RQ S59A kit is an IVD for identification of t 8 21 q22 q22 translocation If used together with REALQUALITY RQ AML1 ETO STANDARD code RQ 60 ST kit it allows the quantification of the number of AML1 ETO transcripts present in the sample normalized to the number of ABL housekeeping gene Such quantification is obtained by the construction of a four point standard curve for AML1 ETO 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 AB2. 16 35127 PADOVA ITALY Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it
3. 8_EN doc 16 www abanaiica Reagent 1 Rx 2X Q Real time Mix 12 5 uL Oligomix ABL 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 uL of cDNA or 5 uL of positive control DNA provided in the kit 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 AML1 ETO 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 paying attention to position it correctly and start the amplification cycle anauitica a 17 RQ S59A 48_EN doc 11 5 QUANTITATIVE ANALYSIS PROTOCOL The quantitative analysis can be performed by using REALQUALITY RQ AML1 ETO STANDARD code RQ 60 ST It is recommended to amplify the samples and standards in duplicate Follow the instructions reported in the previous paragraph to prepare a reaction mix sufficient for the standard curve acquisition and quantification of tested samples both for the AML1 ETO and the ABL gene A negative amplification control must be included in the plate in w
4. ANALITICA ADVANCED BIOMEDICINE User manual REALQUALITY RS AML1 ETO without RT reagents code RQ 859A Kit for identification and quantification of the t 8 21 q22 q22 translocation RQ S59A 48 EN doc 1 PRODUCT INFORMATION 3 1 4 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 7 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 11 9 PRODUCT DESCRIPTION 13 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES 14 11 PROTOCOL 14 11 1 RNA EXTRACTION 14 11 2 RETROTRANSCRIPTION RT FOR cDNA SYNTHESIS 15 11 3 INSTRUMENT PROGRAMMING 15 11 3 1 Creation of thermal protocol 15 11 3 2 Plate Setup 15 Mec ree ire 1 RQ S59A 48 EN doc 11 4 QUALITATIVE ANALYSIS PROTOCOL 11 5 QUANTITATIVE ANALYSIS PROTOCOL 11 6 ANALYSIS AND INTERPRETATION OF QUALITATIVE RESULTS 11 7 ANALYSIS AND INTERPRETATION OF QUANTITATIVE RESULTS 16 18 11 8 NORMALIZATION AND QUANTIFICATION OF MINIMAL RESIDUAL DISEASE 22 11 9 TROUBLESHOOTING 12 13 13 1 13 3 13 4 13 4 13 5 13 6 14 DEVICE LIMITATIONS DEVICE PERFORMANCES Analytical specificity Analytical sensitivity linearity Reproducibility Diagnostic specificity Diagnostic sensitivity Accuracy REFERENCES 14 1 USEFUL WEBSIT
5. ES 15 RELATED PRODUCTS RQ S59A 48 EN doc 22 24 24 24 24 25 26 26 26 27 27 28 anauitica AOVANCED BIOMEDICINE www abanalitica it 1 PRODUCT INFORMATION 1 1 Intended Use The REALQUALITY RS AML1 ETO without RT reagents is an IVD for identification and quantification of the t 8 21 q22 q22 translocation that involves AML1 and ETO genes by amplification of the c DNA in the regions of the AML1 gene on chromosome 21q22 and of the ETO gene on chromosome 8422 If used together with the REALQUALITY RQ AML1 ETO STANDARD code RQ 60 ST kit it allows the quantification of the AML1 ETO transcript 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 samples This in vitro diagnostic test is an auxiliary device for diagnosis and monitoring of clinical pathologies as the Acute Myeloid Leukaemia AML It is recommended to use this kit as indicated in the instructions herein This manual refers to the following product REALQUALITY RS AML1 ETO without RT reagents Kit for identification and quantification of the t 8 21 q22 q22 translocation by Real time PCR This product is in accordance with 98 79 CE Directive Annex 111 regarding the in vitro medical diagnostic devices IVD CE mark Contains all the reagents needed for Real time amplification Code Product PKG RQ S59A 48 REALQUALITY RS AML1
6. ETO 48 test without RT reagents RQ S59A 96 REALQUALITY RS AML1 ETO 96 test without RT reagents anauitica 3 RQ S59A 48_EN doc 2 STORE AT 30 20 C TUBE T DESCRIPTION LABEL OR LID COLOUR Mastermix 2X 2X Q Real time Mix 2 350 3X 450 uL 6X 450 uL Magnesium Chloride solution MgCl 1x 50 uL 1X75 ul 1 X 150 uL Primer and probe Mix AML1 ETO Oligomix Purple 1 x 27 uL 2 x 27 uL 4 x 27 uL Primer and probe Mix ABL Oligomix Blue 1x27 uL 2 x 27 uL 4 x 27 uL STORE AT 2 8 TUBE T DESCRIPTION LABEL OR LID COLOUR AML1 ETO AML1 ETO translocation POSITIVE Purple 1x30 uL 1 x 60 uL 1x110 uL positive control CONTROL ABL POSITIVE ABL positive control Blue 1x30 uL 1x60 uL 1x110 uL CONTROL RQ S59A 48_EN doc a www abanalitica it 3 STORAGE AND STABILITY OF THE REAGENTS Each component of the kit should be stored according to the directions indicated on the label of each box In particular Box P Store at 30 C 20 C Bag Store at 2 C 8 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 t
7. L 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 AML1 ETO amplification because experimental evidences demonstrate that a competition between the two targets can occur in samples with a low number of AML1 ETO 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 prevents contaminations from previous amplifications since it removes residual uracil incorporated in the molecule of single or double stranded DNA NOTE This kit was developed in accordance with the Europe Aga
8. Make sure to perform the extraction of nucleic acids correctly 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 wear 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 anauitica a 23 RQ S59A 48 EN doc 12 DEVICE LIMITATIONS The kit can have reduced performances if e The clinical sample is not suitable for this analysis sampling and or storage error i e blood treated with anticoagulants other than EDTA like heparin etc e The starting samples were not treated as the modality and times indicated in paragraph 10 e The kit was not stored correctly 13 DEVICE PERFORMANCES 13 1 Analytical specificity The analitical specificity of the REALQUALITY RS AML1 ETO without RT reagents code RQ S59A kit is guaranteed by an accurate and specific selection of primers and probes and also by the use of st
9. RETATION OF QUANTITATIVE RESULTS At the end of the reaction view the graph in logarithmic scale Figure 2 Analyze AML1 ETO and ABL graphs and quantification results separately Position the Threshold by choosing the position in which the Correlation anauitica RQ S59A 48_EN doc 20 www abanaiica 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 Delta Rn vs Cycle 1 000 001 Data Della An vs v Detector All 1 000 000 Line Color Well Color v 1 0006 001 r Analysis Settings C Auto Ct 1 000e 002 44 G m k Threshold 0 2 O 1 000e 003 i C Auto Baseline Manual Baseline 1 0006 004 I Start cycle 3 1000805 I L 4 4 Jt ttt End oe 15 Analyze 1 000e 006 Hep 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number Figure 2 Post run data analysis amplification graph displayed in logarithmic scale on the Applied Biosystems 7300 Real T
10. andards and samples e The instrument was not programmed correctly Repeat the amplification taking care 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 making sure to follow the instructions reported in paragraph 11 4 anauitica RQ S59A 48_EN doc 22 wwwabanalitiea t 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 and make sure that they do not undergo any freeze thaw cycle as well Do not use the positive 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
11. antitative protocol define the dilution of the AML1 ETO and ABL standard in the interval from 10 to 10 copies Select and activate the FAM fluorophore and NONE as quencher anauitica a 15 RQ S59A 48_EN doc Pay attention that for the instruments that require it the detection of the fluorescence of the fluorophore ROX corresponds to each 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 pL 11 4 QUALITATIVE ANALYSIS 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 areas 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 H O must be added instead of DNA and when calculating the volume consider an excess of at least one reaction volume Reagent 1 Rx 2X Q Real time Mix 12 5 uL Oligomix AML1 ETO 1 0 uL MgCl 0 5 uL H20 6 0 uL Total Volume 20 0 uL anauitica RQ S59A 4
12. e controls included in the kit and all amplicons in a different area from where reverse transcription and amplification reagents are stored e Organize the work areas in different pre and post PCR units do not share instrument and consumables pipettes tips tubes etc between them e Change gloves frequently e Wash the bench surfaces with 5 Sodium Hypochlorite e Keep the RNA just extracted or that will be stored at 30 C 20 C or 80 C according to the time required between extraction and reverse transcription on ice during reverse transcription preparation anauitica RQ S59A 48_EN doc 6 www abanaltica t 5 SAFETY RULES 5 1 5 2 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 known diagnostic method cannot 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 after decontamination by immerse in a solution of 5 Sodium Hypochlorite 1 vol
13. echanisms 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 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 anauitica a 11 RQ S59A 48_EN doc coo o o 6 PCR Base Line Subtracted CF 7
14. from the treatment beginning A disease relapse means that an amount of therapy anauitica L 9 RQ S59A 48_EN doc resistant residual cells were persistent which characteristics were for a long time unknown since the available analysis techniques had limited sensibility PCR has initiate new possibilities for a more extended and efficient application of MMR monitoring van Dongen et al 1998 Baccarani et al 2006 anauitica RQ S59A 48_EN doc 10 www abanaiica 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 technology 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 m
15. hich the H20 is added instead of cDNA Aliquot 20 uL of the mix in each well of the plate Add 5 uL of cDNA to each well or 5 uL of each quantification standard dilution in the corresponding positions of the plate 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 paying attention to position it correctly and start the amplification cycle 11 6 ANALYSIS AND INTERPRETATION OF QUALITATIVE RESULTS At the end of the reaction view the graph in logarithmic scale Analyze AML1 ETO and ABL graphs and amplification results separately and follow the interpretation pattern as indicated Before analyzing the samples results check the expected results of the positive and negative controls anauitica RQ S59A 48_EN doc 18 www abanaiica RESULT NTERPRETATION ABL Amplification signal Correct ABL amplification positive Control No amplification signal Amplification problems repeat the analysis ABL No amplification signal No contamination negative Control Amplification signal Contamination repeat the analysis RESULT INTERPRETATION AML1 ETO Amplification signal Correct AML1 ETO positive Control amplification No amplification signal Amplification problems repeat the analysis AML1 ETO No amplification signal No contamination negati
16. ime PCR System with SDS software version 1 2 3 Standard Curve 33 193 x alata 32 000 Omit Wels IV Show all standard points for selected detector s 30 000 Detector AMLI Y r Key Ahli Std o X Unknown 26 000 Slope 3 467728 24 000 Intercept 40125543 R2 0 999798 Figure 3 Post run data analysis standard curve on the Applied Biosystems 7300 Real Time PCR System with SDS software version 1 2 3 a 21 RQ S59A 48_EN doc 11 8 NORMALIZATION AND QUANTIFICATION OF MINIMAL RESIDUAL DISEASE The AML1 ETO and ABL standard curves allow to transform the Ct values obtained for unknown samples in AML1 ETO 1 and ABL ABLen copy numbers The normalized copy number NCN of the AML1 ETO transcript is defined as the ratio between the AML1 ETOcn copy number NCNamt1 ETo AML1 ETOcn ABLen The Minimal Residual Disease MRD is expressed as the ratio between the AML1 ETO normalized copy number at the follow up FUP and the AML1 ETO normalized copy number at the time of the diagnosis DX MRD AML1 ETOen AML1 ETOcn ABLen px 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 fup X AML1 ETOpx 11 9 TROUBLESHOOTING Absence of amplification signal for positive controls st
17. inst Cancer guidelines Gabert et al Leukemia 2003 and in accordance with recent international recommendations Branford et al Leukemia 2006 anauitica a 13 RQ S59A 48_EN doc 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES AML1 ETO translocation identification 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 4h time 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 inhibitors i e RLT buffer QUIAGEN 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 manual 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 ext
18. o aliquot the reagents The 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 e The kit must be used only as an IVD and handled by qualified technicians who are well educated and trained in molecular quantitative biology techniques applied to diagnostics e Before starting the kit procedure read carefully and completely the user manual Keep the product 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 anauitica a 5 RQ S59A 48_EN doc e 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 e Prepare the reaction quickly at room temperature or work on ice or ona cooling block In case of any doubts 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 e Use filter tips e Store the biological samples the extracted RNA cDNA and positiv
19. plied Biosystems the kit can be utilized on instruments that use 25 uL of reaction volume and can detect the FAM fluorescence correctly For information on instrument compatibility of the kit please contact AB ANALITICA s technical support 6 3 Materials RQ S59A 48 EN doc 8 Talc free disposable gloves Disposable sterile filter tips range 0 5 10 uL 2 20 uL 10 100 uL 20 200 uL 100 1000 Sterile DNase and RNase free tubes for reverse transcription 96 well plates for Real time PCR and optical adhesive film or 0 1 0 2 mL tubes with optical caps anauitica sitet ho www abanalitica it 7 INTRODUCTION The study of leukemia and lymphoma initiate the comprehension of the cellular and molecular mechanisms at the basis of many neoplastic pathologies by means of easy availability and sampling of leukemic malignant cells present in the blood unlike the ones present in solid tumors that are sometimes difficult to be collected without using invasive samplings techniques The t 8 21 q22 q22 translocation leads to the fusion between AML 1 gene on the 21q22 chromosome and ETO gene on the 8q22 chromosome the break point in the AML1 gene is situated between exon 5 and exon 6 while the one in ETO gene is upstream of exon 2 The resulting fusion protein is constitute by the N terminal domain of the DNA binding of AML1 an essential transcription factor for the haematopoiesis and about all the ETO protein that work
20. raction 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 anauitica RQ S59A 48_EN doc 14 wwwabanalitiea t 11 2 RETROTRANSCRIPTION RT FOR cDNA SYNTHESIS The product was validated using the RevT Kit see paragraph 18 Related product this kit uses a reverse transcription reaction which is in accordance with the Europe Against Cancer guidelines Please follow the instructions reported in the user manual of RevT Kit code 06 01 in particular at the end of reverse transcription reaction please dilute the obtained cDNA 50 ng uL equivalent RNA to a Vein 50 pL adding therefore 30 uL of sterile water 20 ng uL equivalent RNA 11 3 INSTRUMENT PROGRAMMING 11 3 1 Creation of thermal protocol Set the following thermal profile Cycle Repeats Step UNG Activation 1 1 1 2 00 50 0 Taq Activation 2 1 1 10 00 95 0 io 3 45 1 00 15 95 0 2 600 Fluorescence collection step 11 3 2 Plate Setup 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 For the qu
21. ringent amplification conditions Moreover the alignment of primers and probes in the most important databanks shows the absence of non specific pairing 13 2 Analytical sensitivity detection limit The analytical sensitivity limit of REALQUALITY RS AML1 ETO without RT reagents 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 13 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 AML1 ETO and for ABL a linear response for all the panel point 2 gt 0 99 The results of the analysis are reported in Table 1 anauitica RQ S59A 48_EN doc 24 wwwabanalitiea t 13 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 replicates 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 AML1 ETO and for ABL is reported in Table 1 The last point of the quantification standard 20 transcript copies uL corresponding to 100 transcript copie
22. s as a co repressor for a variety of transcription factors The t 8 21 q22 q22 translocation is a rearrangement usually linked to the Acute Myeloid Leukemia AML in particular it is reported in almost 15 of all AML and almost 4096 of FAB M2 subtype of AML AML incidence is approximately 3 5 cases per 100 000 people per year AML can manifest at all ages but the frequency increases in older people Molecular analysis of t 8 21 q22 q22 translocation is very important since this rearrangement is linked to a positive prognosis and in particular to a good response to some therapeutic agents as cytosine arabinoside Bloomfield et al Cancer Res 1998 The translocation detection can be done at the molecular level with a RT PCR which consists of total RNA extraction from starting samples its retrotranscription in CDNA and then amplification of the regions of interest This detection gives useful information for diagnosis and prognosis of these types of leukemia but first of all it allows the monitoring of the Minimal Residual Disease MRD which has important repercussion on therapy MRD is the number of neoplastic cells which is below the identifiable level with standard cytomorphologic techniques present in the organism of the patient affected by leukemia during the different phases of chemotherapy Even if an aggressive chemotherapy makes progress in leukemia treatment a significant percentage of cases relapse at different time point
23. s 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 AML1 ETO and for 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 5 5 transcript AML1 ETO T copies reaction 96 positivity 100 positivity Linear Range transcript copies reaction AML1 ETO ABL Intra assay Variability AML1 ETO ABL Inter assay Variability AML1 ETO anauitica a 25 RQ S59A 48_EN doc 13 4 Diagnostic specificity A significant number of samples negative for AML1 ETO translocation were tested simultaneously with the REALQUALITY RS AML1 ETO without RT reagents kit and another CE IVD or reference method From the obtained results the diagnostic specificity of this device was calculated to be 97 37 13 5 Diagnostic sensitivity A significant number of samples positive for the AML1 ETO translocation were tested simultaneously with the REALQUALITY RS AML1 ETO without RT reagents kit and another CE IVD or reference method From the obtained results the diagnostic sensitivity of this device was calculated to be 100 13 6 Accuracy This value
24. ume of 5 Sodium Hypochlorite solution 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 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 anauitica Tupa d a www abanalitica it 7 RQ S59A 48_EN doc 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 6 2 Reagents Reagents for density gradient separation of mononucleate cells Ficoll RNA extraction reagents RevT Kit cod 06 01 per la reazione di retrotrascrizione vedi paragrafo 18 Prodotti correlati Dnase and Rnase free sterile water Distilled water REALQUALITY RQ AML1 ETO STANDARD code RQ 60 ST for quantitative analysis Instruments 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 Micropipettes range 0 5 10 uL 2 20 uL 10 100 pL 20 200 uL 100 1000 Microcentrifuge max 12 14 000 rpm Plate centrifuge optional Thermalcycler for reverse transcription Real time amplification instrument The kit was standardized on Applied Biosystems 7500 Fast Dx 7300 StepOnePlus Real Time PCR System Ap
25. ve Control Amplification signal Contamination repeat the analysis anauitica a 19 RQ S59A 48_EN doc RESULTS INTERPRETATION sample Amplification signal Amplificable sample ABL amplification ot No amplification signal Sample n suitable for amplification RNA degradation reverse transcription error repeat the RT if again no amplification occurs repeat the RNA extraction Sample Amplification signal AML1 ETO translocation AML1 ETO positive sample amplification No amplification signal AML1 ETO translocation negative sample NOTE When analyzing duplicates one positive well is sufficient to detect a positive sample while both the wells must be negative to diagnose a negative sample for translocation For ABL values samples with ABL Ct corresponding to a copy number that is inferior to the minimum limit of the linearity range see paragraph 13 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 AML1 ETO copy number it allows to be sure that the obtained results is correct and to exclude the possibility that a low copy number of AML1 ETO is due to low cells number in the samples 11 7 ANALYSIS AND INTERP
26. was calculated by the number of correct amplifications over the total number of executed amplifications The REALQUALITY RS AML1 ETO without RT reagents device has an accuracy of 98 33 anauitica RQ S59A 48_EN doc 26 www abanaiica i 14 REFERENCES Baccarani M Saglio G Goldman J et al Blood 15 108 6 1809 20 2006 Bloomfield CD Lawrence D Byrd JC et al Cancer Res 58 4173 4179 1998 Branford S Cross NC Hochhaus A et al Lukemia 20 11 1925 30 2006 Gabert J Beillard E et al Leukemia 17 12 2318 57 2003 Saiki RK S Scharf F Faloona KB Mullis GT Horn HA Erlich and N Arnheim Science 230 1350 1354 1985 Van der Velden VH et al Leukemia 17 1013 1034 2003 van Dongen JJ et al Lancet 352 1731 1738 1998 14 1 Useful websites 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 anauitica 27 RQ S59A 48 EN doc 15 RELATED PRODUCTS REALQUALITY RQ AML1 ETO STANDARD Ready to use quantification standard for t 8 21 q22 q22 translocation transcript Code Product PKG RQ 60 ST REALQUALITY 4 x 60uL AML1 ETO RQ AML1 ETO STANDARD 4 x 60uL ABL anauitica www abanalitica it RQ S59A 48 EN doc 28 ANALITICA ADVANCED BIOMEDICINE www abanalitica it AB ANALITICA srl Via Svizzera
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