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1. Threshold Auto 0 1 Auto Baseline Show Threshold Baseline Start Well Target amp Baseline End Well B Target 4 Analysis Summary Total Wells in Plate 96 Wells SetUp 56 Wells Omitted Manually 0 Wells Flagged 24 Wells Omitted by Analysis 0 Samples Used 17 Targets Used 2 Fig 2 Post run data analysis PML RARa bcr3 amplification graph in logarithmic scale viewed on su Applied Biosystems StepOnePlus Real time PCR System con software version 2 1 Standard Curve lt Plot Settings Target pcne R3 EAC Plot Color Target Save current settings as the default Standard Curve 10 20 100 200 1000 10000 100000 10000 Quantity Target BCR3 EAC Slope 3 312 Y Inter 39 676 p 0 999 Eff 100 42 ge Analysis Summary Total Wells in Plate 96 Wells SetUp 56 Wells Omitted Manually 0 Wells Flagged 24 Wells Omitted by Analysis Samples Used 17 Targets Used 2 Fig 3 Post run Analysis of the data standard curve viewed on su Applied Biosystems StepOnePlus Real time PCR System con software version 2 1 RQ S65 48 EN doc 20 www abanalitica it 12 NORMALIZATION AND QUANTIFICATION OF MINIMAL RESIDUAL DISEASE The PML RARa bcr3 and ABL standard curves allow to transform the Ct values obtained for unknown samples in PML RARa bcr3 PML RARa bcr3 and ABL ABLen copy numbers The normalized copy nu
2. RARa transcripts and sometimes it heavily disadvantages the amplificationof the specific translocation 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 A ready to use mastermix is provided with all the reagents needed except for the oligomix The dUTP UNG system in the mix allows to prevent contaminations from previous amplification being able to remove uracil residues in the single or double stranded DNA molecules NOTE This kit was developed in accordance with the Europe Against Cancer guidelines Gabert et al Leukemia 2003 and in accordance with recent international recommendations Branford et al Leukemia 2006 RQ S65 48 EN doc 12 Qamaumica worm aba naita It 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES The search of the PML RARa bcr3 translocation is performed starting from whole peripheral or bone marrow blood Sample collection should 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 RT PCR reaction Fresh blood can be stored at 2 8 if processed in 4h time after the collection the mononucleate cells must be separated by density gradient centrifugation Ficoll reagent not included in the kit The RNA can
3. RQ S65 48 EN doc 24 Qamaumica www 15 3 Reproducibility In order to determine the inter assay variability variability of the replicates of the same sample in the same run a 50 copy transcript uL of the quantification standard corresponding to a final quantity of 250 transcript copies reaction was amplified in 8 replicates in the same run The intra assay coefficient variability of the method referring to the threshold cycle Ct for RS PML RARa bcr3and ABL is reported in Table 1 In order to calculate the inter assay variability variability of the replicates of the sample in different runs the results obtained from the amplification of the last point of the quantification standard 20 transcript copies pL corresponding to 100 transcript copies reaction conducted in duplicates in at least 3 consecutive runs is considered adequate For each run the variable coefficient was calculated from the Ct of the samples The inter assay variable coefficient for RS PML RARa bcr3 and ABL was calculated from the average of the variable coefficients in each experiment performed as reported in Table 1 ABI 7500 Table 1 Detection Limit Linear Range copies rxn Intra assay Variability Inter assay Variability amend oe www abanahitica it copies rxn PML RARa bcr3 Fast Dx 5 100 positivity ABI 7300 5 95 8 positivity StepOne Plus
4. 2 20 uL 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 adhesive optical film or 0 1 0 2 mL tubes with optical caps RQ S65 48 EN doc 8 www 7 INTRODUCTION Silinmis THanksThe of Of study pathologies lymphoma patients Research on those types of cancer has eem the way to understand the cellular and molecular mechanisms underlying also many Silinmi Those thanks to the availability and samples other neoplastic diseases where cell samples are much harder to obtain or require invasive procedures e g solid tumors Acute Promyelocytic Leukemia APL is a subtype of Acute Myeloid Leukemia AML with a M3 cytomorphology that constitutes 10 1596 of all AML cases In more than 90 of patients the APL is associated with the translocation also called Tui pem Rae gene Jocated in region 22 or 24 UT on nomenclature of the long arm of chromosome 15 and the RARa gene retinoic acid receptor Q in region 21 of the long arm of chromosome 17 The breaking point of the Silinmis of leukemic malignant cells in the blood unlike the solid tumors ones that are sometimes difficult to collect without using invasive samplings J La traslocazione t 15 17 q22 q21 frequentemente associata pi del 9096 dei casi alla Leucemia Acuta Pro
5. 5 100 positivity ABL 10 10 PML RARa bcr3 ABL 0 508 5 10 0 633 0 345 PML RARa ber3 ABL 0 453 0 916 0 799 0 669 0 633 0 454 PML RARa ber3 1 001 25 0 740 0 608 RQ S65 48_EN doc 15 4 Diagnostic specificity A significant number of samples negative for PML RARa bcr3 translocation was tested simultaneously with the REALQUALITY RS PML RARa bcr3 kit and another CE IVD or reference method The obtained results indicate that the diagnostic specificity of this device is 100 15 5 Diagnostic sensitivity A significant number of positive samples for the PML RARa bcr3 translocation were tested simultaneously with the REALQUALITY RS PML RARa bcr3 kit and another CE IVD or reference Real Time method The obtained results were positive for all of the tested samples allowing to calculate a diagnostic sensitivity of 100 15 6 Accuracy This value was calculated by the number of correct amplifications over the total number of executed amplifications The REALQUALITY RS PML RARa bcr3 device has an accuracy of 10096 RQ S65 48 EN doc 26 Qamaumica wore 16 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
6. The reagents should not go undergo more than two freeze thaw cycles If a small number of samples are tested in each batch it is recommended to aliquot the reagents Oligomix and ROX contain fluorescent molecules it is recommended to store these reagents far from any light source 4 PRECAUTIONS FOR USE e The kit must be used only as an IVD and handled by qualified technicians who are educated and trained in molecular biology techniques applied to diagnostics e Before starting the kit procedure read carefully and completely the instruction manual e Keep the product away from heating sources and the direct light e Do not use any part of the kit if over the expiration date In case of any doubt about the storage conditions box integrity or method application contact AB ANALITICA technical support at laboratorio abanalitica it In the amplification of nucleic acids the user has to take the following special precautions e Use filter tips Qanaurtica 5 RQ S65 48_EN doc www abanahitica i e 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 e Organize the work space in different pre and post PCR units do not share consumables pipettes tips tubes etc between them e Change the gloves frequently e Wash the bench surfaces with 5 sodium hypochlorite e Keep the RNA
7. on ice during reverse transcription assembly The RNA can be extracted or stored at 30 C 20 C or at 80 C according to the time between extraction and reverse transcription e Thaw the reagents prior to use once thawed mix the solutions well by inverting the tubes several times do not vortex then centrifuge briefly RQ S65 48 EN doc 6 wwa 5 SAFETY RULES 5 1 General safely rules e Wear disposable gloves to handle the reagents and the clinical samples and wash the hands at the end of work e Do not pipet by mouth e Since no known diagnostic method can assure the absence of infective agents it is a good rule to consider every clinical sample as potentially infectious and handle it as such e All the devices that get directly in touch with clinical samples should be considered as contaminated and disposed 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 e Clinical samples materials and contaminated products should be disposed after decontamination by immersion in a solution of 5 Sodium Hypochlorite 1 volume of 5 Sodium Hypochlorite solution every 10 volumes of contaminated fluid for 30 minutes OR autoclaving at 121 C at least for 2 hours NOTE do not autoclave solutions containing Sodium Hypochlorite 5 2 Safety rules about the kit The ri
8. to artifacts such as pipetting errors or instrument limits In case in which different instruments are used do not add ROX to the reaction mix and adjust the reaction volume appropriately with sterile water Reagent 1 Reaction 1 Reaction 2X Q Real Time Mix 12 5 uL 12 5 uL Oligo Mix PML RARa bcr3 1 uL 1 uL MgCl 0 5 uL 0 5 uL ROX 0 5 uL H O 5 5 uL 6 uL Total Volume 20 uL 20 uL Reagent 1 Reaction 1 Reaction 2X Q Real Time Mix 12 5 uL 12 5 uL Oligo Mix ABL 1 uL 1 uL MgCl 0 5 uL 0 5 uL ROX 0 5 uL H O 5 5 uL 6 uL Total Volume 20 uL 20 uL Mix by inverting the tube in which the mix was prepared several times Then centrifuge briefly Pipet 20 uL of the mix on the bottom of each well on the plate Add 5 uL of cDNA to each well or 5 uL of positive control DNAs included in the kit in the correct positions on the plate RQ S65 48 EN doc 16 Qamaurtica Always amplify a negative control together with the samples to be analyzed add sterile water to the amplification mix instead of cDNA both for the PML RARa bcr3 and the ABL mix Hermetically seal the plate by using the optic adhesive film and the appropriate sealer Make sure 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 QUANTITATIVE ANALYSIS PROT
9. 2 2011 10 45 00 Sayfa 9 5 Silinmis PT Andrej Mantei 09 12 2011 10 45 00 Sayfa 9 6 Silinmis pml Andrej Mantei 09 12 2011 10 45 00 Sayfa 9 6 Silinmis instead Andrej Mantei 09 12 2011 10 46 00 Sayfa 9 6 Silinmis happen Andrej Mantei 09 12 2011 10 46 00 Sayfa 9 6 Silinmis possible Andrej Mantei 09 12 2011 10 46 00 Sayfa 9 6 Silinmis isoform Andrej Mantei 09 12 2011 10 47 00 Sayfa 9 6 Silinmis respectively Andrej Mantei 09 12 2011 10 47 00 Sayfa 9 6 Silinmis Andrej Mantei 09 12 2011 10 47 00 Sayfa 9 6 Silinmis Sayfa 9 6 Silinmis Sayfa 9 7 Silinmis e derived Andrej Mantei Andrej Mantei Andrej Mantei 09 12 2011 10 47 00 09 12 2011 10 47 00 09 12 2011 10 48 00 Sayfa 9 7 Silinmis the Andrej Mantei 09 12 2011 10 48 00 Sayfa 9 7 Silinmis Andrej Mantei 09 12 2011 10 48 00 Sayfa 9 7 Silinmis blocked Andrej Mantei 09 12 2011 10 49 00 Sayfa 9 7 Silinmis working as a differentiation driving factor Andrej Mantei 09 12 2011 10 49 00 Sayfa 9 8 Silinmis detection can be done Andrej Mantei 09 12 2011 10 50 00 Sayfa 9 8 Silinmis with a Andrej Mantei 09 12 2011 10 50 00 Sayfa 9 8 Silinmis RT PCR which consists of a Andrej Mantei 09 12 2011 10 51 00 Sayfa 9 8 Silinmis its retro transcription And
10. 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 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 Qanaurtica 27 RQ S65 48_EN doc 18 RELATED PRODUCTS REALQUALITY RQ PML RARa bcr3 STANDARD Quantified and ready to use quantification standard for the transcript of the t 15 17 q22 q21 translocation in the bcr3 variant Code Product PKG RQ 66 ST REALQUALITY 4 x 60 uL PML RARa bcr3 RQ PML RARa bcr3 STANDARD 4 x 60 uL ABL REALQUALITY RS PML RARa bcr1 Kit for the identification and quantification of the t 15 17 q22 q21 translocation in the bcr1 variant by Real Time PCR Code Product PKG RQ S63 REALQUALITY RS PML RARa 48 96 test bcr1 RQ S65 48 EN doc 28 Qamaumica 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 Sayfa 9 1 Silinmis THanksThe Andrej Mantei 07 12 2011 17 42 00 Sayfa 9 1 Silinmis study of Andrej Mantei 07 12 2011 17 44 00 Sayfa 9 1 Silinmis of Andre
11. 68 Y 3 338 X 38 8 o Unknowns PCR Efficiency 99 3 Standards Threshold Cycle Ie C OO R RO 3 4 5 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 11 RQ S65 48_EN doc 9 PRODUCT DESCRIPTION REALQUALITY RS PML RARa bcr3 kit code RQ S65 allows the identification of t 15 17 q22 q21 translocation in the bcr3 variant If used with the REALQUALITY RQ PML RARa bcr3 STANDARD code RQ 66 ST product the kit allows the absolute quantification of the number of PML RARa bcr3 transcripts in the sample normalized compared to the number of ABL housekeeping gene transcripts Such quantification is obtained by making a standard curve with 4 points for PML RARa bcr3 and in parallel a standard curve with 4 points for ABL The amplification of ABL gene is made separately from the one for PML RARa because experimental evidence demonstrates that a competition between the two targets can occur in samples with a low number of PML
12. ANALITICA ADVANCED BIOMEDICINE www abanalitica it User manual REALQUALITY RS PML RARa bcr3 code RQ S65 Kit for the identification and quantification of the t 15 17 q22 q21 translocation bcr3 variant RQ S65 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 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 Retrotranscription RT for the synthesis of cDNA 13 11 3 INSTRUMENT PROGRAMMING 15 11 3 1 Creation of thermal protocol 15 11 3 2 Plate Setup 15 11 4 QUALITATIVE ANALYSIS PROTOCOL 15 11 5 QUANTITATIVE ANALYSIS PROTOCOL 17 11 6 ANALYSIS AND INTERPRETATION OF QUALITATIVE RESULTS 18 11 7 ANALYSIS AND INTERPRETATION OF QUANTITATIVE RESULTS 19 12 13 14 15 15 1 15 2 15 3 15 4 15 5 15 6 16 17 18 NORMALIZATION AND QUANTIFICATION OF MINIMAL RESIDUAL DISEASE21 TROUBLESHOOTING DEVICE LIMITATIONS DEVICE PERFORMANCES Analytical specificity Analytical sensitivity Reproducibility Diagnostic specificity Diagnostic sensitivity Accuracy REFERENCES USEFUL LINKS REL
13. ATED PRODUCTS RQ S65 48_EN doc 22 24 24 24 24 25 26 26 26 27 27 28 Qanauitica ter d sei www abanalitica it 1 PRODUCT INFORMATION 1 1 Intended Use The REALQUALITY RS PML RARa bcr3 is an IVD for the identification and quantification of the t 15 17 q22 q21 translocation in the bcr3 variant by amplification of c DNA in the regions of the genes involved in pml on chromosome 15 and rara on chromosome 17 The kit allows the absolute quantitative analysis of the PML RARa bcr3 transcript present in the sample if used together with the related product REALQUALITY RQ PML RARa bcr3 STANDARD code RQ 66 ST The technique used in this kit is the Real time PCR amplification starting from c DNA obtained by reverse transcription of the RNA extracted from human clinical samples This is an in vitro diagnostic test that can be used as an auxiliary device for detection and monitoring of the Acute Myeloid Leukaemia AML in particular the Acute Promyelocytic Leukemia APL The present manual refers to the following product REALQUALITY RS PML RARa bcr3 Kit for the identification and quantification of the t 15 17 q22 q21 translocation in the bcr3 variant by Real time amplification This product is in accordance with 98 79 CE Annex III Directive regarding the in vitro medical diagnostic devices CE mark Contains all the reagents needed for the Real time amplification Code Product PKG RQ S65 48 REALQUALITY RS P
14. I the RNA jnto cDNA_and specific amplification and quantification of the isoform respectively lt 6 fused region s IN Silinmi e derived the x Ns blocked working as a Detection and analysis of the translocation not only delivers valuable differentiation driving fac p71 information for diagnosis and prognosis of these types of leukemia but above all provides the means to monitor the Minimal Residual Disease Minimal Residual Disease MRD denotes the survival of a number of following Silinmi detection can be done with a RT PCR which consists of a its retro transcription and the ofthe region 8 neoplastic cells the body of a cancer leukemia patient during chemotherapy and or a symptomless remission phase Those cells usually cannot be detected using standard cytomorphologic techniques Although on therapy Silinmi This detection gives useful first of all allows with consequent repercussion 2 9 aggressive chemotherapy has considerably advanced leukemia treatment a significant percentage of patients experience a relapse jn various stages after the treatment Usually this js the result of the presence of therapy resistant residual cells The characteristics of these cells have remained unknown for a Silinmis MRD describes the thatremain the which ca
15. ML RARa bcr3 48 test RQ S65 96 REALQUALITY RS PML RARa bcr3 96 test Qanaurtica 3 RQ S65 48_EN doc 2 KIT CONTENT STORE AT 30 20 C TUBE T DESCRIPTION LABEL OR LID COLOUR Reagents for reverse RT MIX 3x143uL 6x135uL 6x260 uL transcription Reverse transcription RT enzyme 1x17 uL 1x30 uL 2x 30 uL Mastermix 2X 2X Q Real Time mix 2x350uL 3X450uL 6X 450 uL Magnesium Chloride solution MgCl 1x50 pL 1X75 pL 1X 150 uL ROX Reference Dye ROX 1x 30 uL 1X60 uL 2X 60 uL PML RARa bcr3 Primer and probe Mix Oligomix Green 1x27 uL 2x27 uL 4 x 27 uL Primer and probe Mix ABL Oligomix Blue 1x27 uL 2x27 uL 4x 27 uL STORE AT 2 8 TUBE T DESCRIPTION LABEL OR LID COLOUR PML RARa bcr3 m POSITIVE Green 1 1x60pL 1x110pL anslocation positive co CONTROL ABL POSITIVE ABL positive control CONTROL Blue 1x30 uL 1x60 uL 1x110 uL NALITICA RQ S65 48 EN doc ww 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 the single boxes In particular Box P Store at 30 C 20 C Bag Store at 2 8 When stored at the recommended temperature all test reagents are stable until their expiration date The 2X Q Real Time Mix and Oligomix reagents are sensitive to the physical state variations
16. OCOL The quantitative analysis can be performed by using the related product REALQUALITY RQ PML RARa bcr3 STANDARD code RQ 66 ST It is recommended to amplify both the samples and the standards in duplicates 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 PML RARa bcr3 and the ABL gene A negative amplification control must be included on the plate in which the H20 is added instead of cDNA Aliquot 20 uL of the mix on the bottom of each well on the plate Add 5 uL of cDNA to each well or 5 uL of each quantification standard dilution in the corresponding positions on the plate Hermetically seal the plate by using the optic adhesive film and the appropriate sealer Make sure 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 Qanaurtica 17 RQ S65 48 EN doc 11 6 ANALYSIS AND INTERPRETATION OF QUALITATIVE RESULTS Check the reaction graph when the reaction has ended Analyze PML RARa bcr3 and ABL graphs and amplification results separately And follow the interpretation pattern as indicated Before analyzing the samples results check the expected positive and negative controls results RESULT INTERPRETATION ABL Amplificatio
17. a 9 10 Silinmis ence Andrej Mantei Andrej Mantei 09 12 2011 11 06 00 09 12 2011 11 06 00 Sayfa 9 10 Silinmis of a number of Andrej Mantei 09 12 2011 11 06 00 Sayfa 9 10 Silinmis whose Andrej Mantei 09 12 2011 11 07 00 Sayfa 9 10 Silinmis sensibility Andrej Mantei 09 12 2011 11 08 00 Sayfa 9 10 Silinmis analysis Andrej Mantei 09 12 2011 11 08 00
18. als Positive controls standards were stored inappropriately and have decayed Make sure to store the positive controls standards appropriately at 2 8 and that these reagents do not undergo any freeze thaw cycle as well Do not use the positive controls standards beyond the expiry date The reaction mix does not function correctly Make sure to store the 2X Q Real Time mix and oligomix appropriately at 20 C 30 C Avoid unnecessary freeze thaw cycles Delayed or no ABL amplification signal in the samples The extracted RNA is not suitable for amplification or a problem may have occurred during the reverse transcription reaction and the amplification reaction is inhibited Make sure to perform the extraction of nucleic acids correctly f an extraction system uses Ethanol wash steps make sure no ethanol residual remains in the DNA sample Use the extraction systems suggested in paragraph 10 1 during the reverse transcription reaction check visually the deposition of the Reverse Transcriptase enzyme in the tube by RQ S65 48 EN doc 22 Qamaumica worm aba nakta It observing the drop formed by the enzyme on the tube wall after its addition to the mix then centrifuge briefly Follow the standard procedures for minimization of the RNA degradation use the RNase free plastic lab ware and work on ice during the reverse transcription reaction For any further problems contact AB ANALITICA s tec
19. am the following thermal profile 1 cycle 70 10 min Next put the tubes on ice immediately for at least 5 minutes Add 14 5 uL of RT mix and 0 5 uL of RT Enzyme mix by pipetting centrifuge briefly and incubate in a thermalcycler programmed as below 20 10 min 1 cycle 42 C 45 min 99 C 3 min 4 C 5 min Once the cycle ends add 30 of sterile water to each retrotranscribed sample The diluted cDNA can be stored in a fridge for short period of time maximum one week or at 20 C 30 C for long periods of time N B this cDNA can be used both for the amplification of the PML RARa bcr3 translocation and ABL housekeeping gene Further the same cDNA can be used also be used for the test of the PML RARa bcr1 translocation by using the correlated product REALQUALITY RS PML RARa bcr1 code RQ S63 RQ S65 48 EN doc 14 Qamaumica worm aba naita It 11 3 INSTRUMENT PROGRAMMING 11 3 1 Creation of thermal protocol Set the following thermal profile Cycle Repeats Step Time C UNG Activation 1 1 1 2 00 50 0 Taq Activation 2 1 1 10 00 95 0 3 45 1 00 15 95 0 cycles 2 010 9600 Fluorescence collection step 11 3 2 Plate Setup Mark the negative control NTC standard STD and sample Unknown position on the grid corresponding to the new plate in the same position as loaded on the plate and identify each sample with a nam
20. be extracted immediately from the pellet containing the lymphocytes otherwise the cell pellet may be conserved at 80 until the RNA extraction better if preserved in a buffer containing RNAse inhibitors ex RLT buffer QIAGEN or Trizol 11 PROTOCOL 11 1 RNA extraction The product was validated using the RNeasy Mini kit QIAGEN However it is possible to use any extraction method provided that it allows the isolation of pure and integral RNA Please follow the instructions below regarding the quantity of RNA to be used for the reverse transcription reaction about 1g 11 2 Retrotranscription RT for the synthesis of cDNA Attention before starting the reverse transcription procedures it is recommended to use an ice container and to thaw one or more RT mix aliquotes depending on the number of analyzed samples Once thawed the RT mix must be mixed well by inverting the tube a number of times do not vortex then centrifuged briefly and stored on ice until use 13 RQ S65 48 EN doc www abanahitica it For each sample use a sterile DNase and RNase free tube and add see paragraph 6 3 Extracted RNA 5 yL 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 H20 Insert the tubes in the thermalcycler and progr
21. d 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 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
22. e NOTE It is recommended to amplify both the samples and positive controls standards in duplicates For the quantitative protocol define the dilutions of the PML RARa bcr3 standards and the ABL standards in a range from 10 copies to 10 copies Select and activate the FAM fluorophore and set the quencher to NONE Pay attention that the ROX fluorescence detection is activated in each position if required by the instrument used Set the reaction volume to 25 uL if and when needed 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 Take care to work shaded from the direct light as much as possible Prepare a master mix of an appropriate volume that must be sufficient for all the samples to be processed for the positive controls and the negative control in which the H2O is added instead of DNA when calculating the Qanaurtica 15 RQ S65 48_EN doc ee er volume consider an excess of at least one reaction volume as indicated below It is recommended to amplify both controls standards in duplicates the samples positive ROX is an inert dye whose fluorescence does not change during the amplification reaction on instruments that allow its use Applied Biosystems Stratagene etc it allows to normalize the well to well differences due
23. entific 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 PML RARa bcr3 1 copy number it allows the obtained result to be sure and it excludes the possibility that a low copy number of PML RARa bcr3 is due to the low cell number in the starting sample 11 7 ANALYSIS AND INTERPRETATION OF QUANTITATIVE RESULTS Check the reaction graph when the reaction has ended Analyze the PML RARa bcr3 and ABL graphs and quantification results separately View in the logarithmic scale Fig 2 and position the threshold in order to obtain a correlation coefficient R and slope of the standard curve closest to the following respective values 1 and 3 33 Fig 3 The results are acceptable with a amplification efficiency within the 85 110 range slope about 3 75 3 10 and a correlation coefficient not less than 0 990 19 RQ S65 48_EN doc www abanahitica it Amplification Plot Plot Settings Plot Type ARn vs Cycle z Graph Type Log Plot Color rarget 2 Save current settings as the default v P EI Amplification Plot 10 T 1 0 1 0 01 T 0 0001 0 00001 0 000001 2 4 a 10 12 14 16 18 20 22 24 260 28 30 32 34 36 38 40 42 44 Cycle Options Target BCR3 EAC
24. hnical support at laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 Qanaurtica 23 RQ S65 48 EN doc 14 DEVICE LIMITATIONS The kit can have reduced performances if e The clinical sample is not suitable for this analysis 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 analytical specificity of the REALQUALITY RS PML RARa bcr3 kit code RQ S65 is guaranteed by an accurate and specific selection of primers and probe and also by the use of the 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 PML RARa bcr3 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 Linear range It was calculated using a dilution panel of the quantification standard The analysis of the data obtained with a linear regression demonstrated that the dose present for the RS PML RARa bcr3 and ABL give linear responses to all the points in the panel R gt 0 99 The results are reported in Table 1
25. j Mantei 09 12 2011 10 17 00 Sayfa 9 1 Silinmis pathologies Andrej Mantei 09 12 2011 10 16 00 Sayfa 9 2 Silinmis Andrej Mantei 09 12 2011 10 18 00 of leukemic malignant cells in the blood unlike the solid tumors ones that are sometimes difficult to collect without using invasive samplings La traslocazione t 15 17 q22 q21 frequentemente associata piu del 90 dei casi alla Leucemia Acuta Promielocitica APL un sottotipo di Leucemia Mieloide Acuta AML con citomorfologia M3 Le APL costituiscono il 10 15 dei casi di AML The t 15 17 q22 q21 Sayfa 9 2 Silinmis comes Andrej Mantei 09 12 2011 10 28 00 Sayfa 9 2 Silinmis from the Andrej Mantei 09 12 2011 10 28 00 Sayfa 9 2 Silinmis between the pml Andrej Mantei 09 12 2011 10 29 00 Sayfa 9 3 Silinmis situated Andrej Mantei 09 12 2011 10 29 00 Sayfa 9 3 Silinmis on Andrej Mantei 09 12 2011 10 35 00 Sayfa 9 3 Silinmis q22 Sayfa 9 3 Silinmis fara Sayfa 9 4 Silinmis situated on Andrej Mantei Andrej Mantei Andrej Mantei 09 12 2011 10 35 00 09 12 2011 10 31 00 09 12 2011 10 35 00 Sayfa 9 4 Silinmis q21 Andrej Mantei 09 12 2011 10 36 00 Sayfa 9 4 Silinmis rara Andrej Mantei 09 12 2011 10 44 00 Sayfa 9 5 Silinmis located Andrej Mantei 09 12 2011 10 45 00 Sayfa 9 5 Silinmis the Andrej Mantei 09 1
26. mber NCN of the PML RARa bcr3 transcript is defined as the ratio between the PML RARa bcr3 en and ABLen copy number NCNpm RARa bcr3 PML RARa bcr3 cN The Minimal Residual Disease MRD is expressed as the ratio between the PML RARa bcr3 normalized copy number at the follow up FUP and the PML RARa bcr3 normalized copy number at the time of the diagnosis DX MRD PML RARa bcr3 cN ABLen rup PML RARa bcr3 cN 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 PML RARa bcr3 px Qanaurtica 21 RQ S65 48 EN doc www abanahitica i 13 TROUBLESHOOTING Absence of positive control standard and sample amplification signal 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 The amplification mix was not prepared correctly Prepare a new amplification mix making sure to follow the instructions given in the paragraph 11 4 The kit was not stored properly or it was used beyond the expiry date Check both the storage conditions and the expiry date reported on the label use a new kit if needed Delay in positive controls standard amplification sign
27. mielocitica APL un sottotipo di Leucemia Mieloide Acuta AML con citomorfologia M3 Le APL costituiscono il 10 1596 dei casi di The t 15 17 q22 q21 comes from the between z 2 RARa gene is inside a region of 15 Kb of intron 2 The breaking point of the gene can be found different regions intron 6 55 of cases L the pml Bi imlendirilmi Yaz tipi talik intron 3 4 0 of cases and exon 6 5 of the cases Each breaking point is Silinmis situated on q22 rara 3 associated with a particular isoform of the PML RARa fusion protein called bcr 1 bcr 3 and bcr 2 respectively Bicimlendirilmis Yaz tipi Italik This fusion protein is a transcriptional repressor that blocks the differentiation 421 Silinmis situated n 4 of hematopoietic stem cells which then remain jn the promyelocyte stage Treatment with ATRA All Trans Retinoic Acid as a differentiation drivin Bicimlendirilmis Yazi tipi Italik factor is a very effective therapy for APL Silinmig located ihe S Bicimlendirilmis Yaz tipi The translocation can be detected at the molecular level using a method that ok involves extraction of total RNA of the starting sample reverse transcription Silinmi pm instead happen possible of the R
28. n 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 PML RARa bcr3 Amplification signal Correct PML RARA BCR3 Positive Control amplification No amplification signal Amplification problems repeat the analysis PML RARa bcr3 No amplification signal No contamination Negative Control Amplification signal Contamination repeat the analysis RQ S65 48 EN doc 18 Qamaumica www abanalitica it RESULT INTERPRETATION Amplification signal Sample suitable for amplification amplification No amplification signal Sample suitable for ipic p degradation reverse transcription error repeat the RT if again no amplification occurs repeat the RNA extraction Sample Amplification signal PML RARA BCR3 translocation PML RARa bcr3 positive sample amplification No amplification signal PML RARA BCR3 translocation 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 the copy n that is lt than the minimum limit of the linearity range see paragraph 15 Device performances must be excluded from the analysis The International sci
29. rej Mantei 09 12 2011 10 51 00 Sayfa 9 8 Silinmis and the following Andrej Mantei 09 12 2011 10 51 00 Sayfa 9 8 Silinmis of the regions of interest Andrej Mantei 09 12 2011 10 53 00 Sayfa 9 9 Silinmis This detection gives useful Andrej Mantei 09 12 2011 10 55 00 Sayfa 9 9 Silinmis first of all allows Andrej Mantei 09 12 2011 10 55 00 Sayfa 9 9 Silinmis with consequent repercussion on therapy Andrej Mantei 09 12 2011 10 58 00 Sayfa 9 10 Silinmis MRD describes Andrej Mantei 09 12 2011 10 58 00 Sayfa 9 10 Silinmis the Andrej Mantei 09 12 2011 11 00 00 Sayfa 9 10 Silinmis that remain Andrej Mantei 09 12 2011 11 00 00 Sayfa 9 10 Silinmis which Sayfa 9 10 Silinmis Andrej Mantei Andrej Mantei 09 12 2011 11 03 00 09 12 2011 11 03 00 Sayfa 9 10 Silinmis with Andrej Mantei 09 12 2011 11 03 00 Sayfa 9 10 Silinmis the Andrej Mantei 09 12 2011 11 03 00 Sayfa 9 10 Silinmis cases Andrej Mantei 09 12 2011 11 05 00 Sayfa 9 10 Silinmi at Andrej Mantei 09 12 2011 11 06 00 Sayfa 9 10 Silinmi different Andrej Mantei 09 12 2011 11 05 00 Sayfa 9 10 Silinmi time Andrej Mantei 09 12 2011 11 06 00 Sayfa 9 10 Silinmis A disease relapse Andrej Mantei 09 12 2011 11 06 00 Sayfa 9 10 Silinmis the persist Sayf
30. ses at different time A disease relapse the persist ence of a number of Whose sensibility analysis 10 long time due to the limited sensitivity of techniques available Lately the Bicimlendirilmis Yazi tipi Varsayilan Arial 14 nk Kalin De il RQ S65 48_EN doc monitoring van Dongen et al 1998 Baccarani et al 2006 In the light of what has been said before molecular analysis of the t 15 17 q22 q21 translocation appears very important for diagnosis and es gk OS oa monitoring of APL patients Not only detection but also quantification of the PML RARa transcript proved to be an important tool It has been shown that the amount of PML RARa transcript during the recovery phase is a strong risk indicator for a hematologic relapse Also levels of PML RARa transcript remaining high during remission maintenance therapy and the end of chemotherapy were associated with a high risk of clinical relapse and a negative prognosis Santamaria et al Haematologica 2007 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 contribute
31. sks 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 Qanaurtica 7 RQ S65 48_EN doc 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 Reagents Reagents for density gradient separation of mononucleate cells Ficoll RNA extraction reagents Dnase and Rnase free sterile water Distilled water REALQUALITY RQ PML RARa bcr3 STANDARD code RQ 66 ST for quantitative analysis 6 2 Instruments e Laminar flow cabinet its use is recommended for preparing the amplification mix to avoid contamination it is recommended to use another laminar flow cabinet to add the extracted DNA and standards e Micropipettes range 0 5 10 uL 2 20 uL 10 100 uL 20 200 pL 100 1000 pL e Microcentrifuge max 12 14 000 rpm e Plate centrifuge optional e Thermocycler for reverse transcription e Real time amplification instrument The product was validated on Applied Biosystems 7500 Fast DX 7300 and StepOnePlus Real Time PCR system Applied Biosystems the product is compatible with instruments that can use 25 uL of reaction volume and that can detect the FAM fluorescence correctly For more information regarding the compatibility of the kit with other commercial instruments please contact AB ANALITICA s technical assistance 6 3 Materials e Talc free disposable gloves e Disposable sterile filter tips range 0 5 10 uL
32. target nucleic RQ S65 48 EN doc 10 worm aba naita It Silinmi The Silinmi opened Silinmi application on MMR Silinmi Therefore The Silinmi is Silinmis the Silinmi the Silinmis In particular the positivity of Silinmi Silinmi is associated with different levels of risk for reoccurrence During the recovery phase Silinmi positivity Silinmi is A klama AM1 a following hematological relapse A klama AM2 Silinmi predictive indicator for successive homological reoccurrences during the maintaining therapy until the end of the chemotherapy treatment the positivity of PML RARa is associated gt Silinmis reoccurrence 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 A aa dar SZ PCR Base Line Subtracted CF RFU 7 0 2 4 6 B 10 12 14 16 18 20 22 2f 28 30 32 34 36 38 40 42 44 46 cde Correlation Coefficient 0 999 Slope 3438 Intercept 28 7

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