USER MANUAL
Contents
1. BanALITICA pag 7 04 60R 50 8033622780731 EN doc 6 PREPARATION OF THE REAGENTS Preparation of 1 L of 1X TAE buffer e Mix 200 mL of 5X TBE with 800 mL of distilled water BANALITICA Term pag 8 04 60R 50 8033622780731 EN doc 7 INTRODUCTION The lymphoid diseases include malignant lymphoma non Hodgkin lymphoma and Hodgkin disease and the chronic lymphoid leukaemia Harris et al 1994 In the diagnosis of these pathologies the molecular techniques Southern blot and Polymerase Chain Reaction are becoming more popular Their applications are in the diagnosis of complete or partial disease remission and in monitoring the minimal residual disease MRD A correct diagnosis of haematological diseases needs a multidisciplinary approach that allows to correlate the traditional morphological evaluations Hematoxylin eosin stain or Wright with a variety of special analysis like immuno histochemical staining molecular and cytogenetic techniques Lymphoid diseases are an heterogeneous group of diseases that result as the consequence of a B and T lymphocytes neoplastic transformation in different phases of their development Their big variety is related to lymphocytes diversity during the different phases of their development and to the huge complexity of the immune system Since the DNA rearrangements take place in great numbers during T cells development T cells tumours are very frequent As for B cells tumours T2. 04 60R 50 8033622780731 EN doc 11 PROTOCOL 11 1 DNA EXTRACTION For DNA extraction anv extraction method can be used provided that it allow to isolate an integral and pure DNA The obtained DNA should be quantified by photometric measures The DNA amount to use in the amplification is 500 ng and if necessary the obtained solution should be diluted Please note that the required volume of extracted DNA to be added in each premix tube is 10 uL For any further information contact AB ANALITICA technical support e mail laboratorio abanalitica it BANALITICA pag 16 04 60R 50 8033622780731 EN doc 11 2 y CHAIN V J REGION DNA AMPLIFICATION 11 2 1 Synthesis of a single strand DNA Add into each MM1 colourless test tube containing 9 9 uL of mix and a wax bead AB Taq 0 1 uL Extracted DNA 5 uL Sterile water 5 uL It is important to include in each experiment a negative polyclonal control to monitor the contamination any genomic DNA of a healthy patient and 10 uL of a monoclonal positive control included in the kit Moreover the introduction of a negative amplification control water instead of DNA will allow to monitor the contaminations The ideal amount of DNA to amplify is 500 ng It is suggested to verify the extracted DNA concentration by photometric measures In particular for DNA extracted from paraffin included specimen it could be necessary to amplify a DNA sample bigger that 5 uL in this
3. 2 04 60R 50 8033622780731 EN doc 1 KITCONTENT BOX P DESCRIPTION LABEL 8 test MM1 monodose premix tubes Colourless T 25 50 10 MM2 monodose premix tubes Green T 25 50 10 Thermostable Taq DNA AB TAQ polymerase 5 U uL Red 1x 25 ul 1x 50 uL 1x10 pl SMALL BAG Plasmid DNA obtained from Positive control monoclonal T cells yTCR Bile 19690 jil Lal TI BOXF Electrophoresis Buffer for sample loading Blu 6X Blue 1x150uL 1x300uL 1x 100 uL Ethidium Bromide solution stat 2 5 mg mL g olla Red 1x80uL l 1x150uL 1x50 uL 8 R 23 68 S 36 37 45 DNA Molecular Weight Marker MW Marker Yellow 1x80 uL 1x150 uL 1x50uL BOX A Agarose for molecular biology AGAROSE 1x10g 1x20g 1x5g Electrophoresis Buffer TRIS Borate EDTA pH 8 00 TBE 50X 1x250mL 1x 500mL 1x 100 mL BanaLimoa pag 3 me 04 60R 50 8033622780731 EN doc 2 STORAGE AND STABILITV 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 20 C Small bag store at 207C Box F store at a 2 8 C Box A store at 15 25 C When stored at the recommended temperature all test reagents are stable until their expiration date 3 PRECAUTIONS FOR USE e The kit should be handled by investigator qualified through education and training in molecular biology tec
4. false positive results Nevertheless its always recommended to use all the proper amplification controls The MM1 premixes include a wax bead this bead will allow to perform a hot start amplification during ssDNA amplification the Taq DNA polymerase becomes active only after the initial denaturation step when the wax melt will allow its descending in the mix The hot start amplification reduces the possibility of an eventual presence of unspecific amplified products Consumo pag 13 04 60R 50 8033622780731 EN doc 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES 10 1 Peripheral or bone marrow blood During the sample collection follow all the usual sterility precautions Blood should be treated with EDTA Other anticoagulating 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 8 C and processed in a short time if DNA extraction is not performed in a short time the sample must be frozen 10 1 1 Pre treatment of peripheral or bone marrow blood In order to obtain a more appreciable yield in the DNA extraction it is recommended to prepare a buffy coat by centrifuging the whole blood at 3300 x g for 10 min at room temperature Then three different fractions will be distinguishable the plasma is the clear upper fraction the buffy coat is the intermediate fraction containing the concentrated le
5. ANALITICA ADVANCED BIOMEDICINE www abanalitica it USER MANUAL yTCR Kit for the analysis of clonality of V J regions of y chain of T cell receptor TCR REF 04 60A 04 60R 50 8033622780731 EN doc 1 KITCONTENT 2 STORAGE AND STABILITV OF THE REAGENTS 3 PRECAUTIONS FOR USE 4 SAFETV RULES 4 1 General safetv rules 4 2 Safetv rules about the kit 5 MATERIALS REQUIRED BUT NOT PROVIDED 5 1 Reagents 5 2 Instruments 5 3 Materials 6 PREPARATION OF THE REAGENTS 7 INTRODUCTION 8 TEST PRINCIPLE o YNNN 12 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES 10 1 Peripheral or bone marrow blood 10 1 1 Pre treatment of peripheral or bone marrow blood 10 2 Histological samples 10 2 1 Pre treatment of histological samples 11 PROTOCOL 11 1 DNA EXTRACTION 11 2 y CHAIN V J REGION DNA AMPLIFICATION 11 2 1 Synthesis of a single strand DNA 11 2 2 TCR y CHAIN V J REGION ssDNA AMPLIFICATION pag 1 04 60R 50 8033622780731 EN doc 14 14 14 14 15 16 16 17 17 18 BanALITICA 11 3 VISUALIZATION OF THE AMPLIFICATION PRODUCTS 19 11 3 1 Agarose gel electrophoresis 19 11 3 2 Sample loading 19 11 4 INTERPRETATION OF THE RESULTS 21 12 TROUBLESHOOTING 23 13 DEVICE LIMITS 25 14 DEVICE PERFORMANCES 25 14 1 Specificity 25 14 2 Sensitivity 25 15 BIBLIOGRAPHIC REFERENCES 26 16 INFORMATION FOR ORDERS 27 16 1 RELATED PRODUCTS 28 Quum pag
6. LITICA pag 27 04 60R 50 8033622780731 EN doc 16 1 RELATED PRODUCTS yTCR Kit for the analysis of clonality of V J regions of the y chain of T cell receptor TCR cod 04 60R The kit contains the reagents for DNA amplification and positive control Target region T cell receptor gene TCR Cod Prod Pkg 04 60R 25 yTCR 25 tests 03 60R 50 yTCR 50 tests anamnes pag 28 04 60R 50 8033622780731 EN doc 04 60R 50 8033622780731 EN doc ANALITICA AB ANALITICA srl Via Svizzera 16 35127 PADOVA ITALY S Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it
7. LONAL in the range populations 160 bp 190 bp BaNALITICA pag 21 04 60R 50 8033622780731 EN doc Fig 1 396 HR agarose gel electrophoresis after a 5 hours run MW DNA Marker Positive sample for T cells receptor monoclonality Jurkatt cell line PATIENT 1 monoclonal sample PATIENT 2 biclonal sample Negative polyclonal control Negative control of amplification water DNA MW Marker DUBITO BaNALITICA pag 22 04 60R 50 8033622780731 EN doc 12 TROUBLESHOOTING 1 Neither amplification products nor positive control DNA band e TAQ polvmerase was not correctiv added to the premix Use pipets and tips with suitable volumes pipet range 0 2 2 uL Check visually that TAQ polymerase diffuses in the premix this is easy because the enzyme is dissolved in glycerol that has a higher density alternatively check visually the drop of TAQ polymerase put on the tube wall then centrifuge briefly e The thermalcycler was not correctly programmed Check the conformity of the thermalcycler program and the temperature profile in the instruction manual then repeat the amplification with the correct program e The kit doesn t work properly Store the premix TAQ polymerase and positive control at 20 C Avoid repeated freezing thawing of the premix and the reagents 2 Presence of unspecific products out of the respective range after visualisation in agarose gel e The thermalcycler makes tempera
8. banks shows the absence of unspecific alignment Cross reactions with genomic DNA have not been revealed 14 2 Sensitivity The sensitivity of the method was tested with samples in which monoclonal polyclonal Tcells and B cells were mixed The system is able to detect until 3 of monoclonal T cells in a population of polyclonal T cells and until 196 in a population of B cells BanALITICA pag 25 04 60R 50 8033622780731 EN doc 15 BIBLIOGRAPHIC REFERENCES Cossman J Uppenkamp M sundeen J Coupland R Raffeld M Arch Pathol Lab Med 112 117 27 1988 Greer J P Kinney M C Loughran TP T cell and NK Cell Lymphoproliferative disorders Haematology 1 259 286 2001 Harris HL Jaffe ES Stein H Banks PM Chan JKC Clearly ML et al Blood 84 1361 92 1994 Saiki RK S Scharf F Faloona KB Mullis GT Horn HA Erlich and N Arnheim Science 230 1350 1354 1985 Zucchini A Bagli L Fattori P P Ravaioli A Zamai L Papa S Biologi Italiani 9 36 42 2002 BANALITICA pag 26 04 60R 50 8033622780731 EN doc 16 INFORMATION FOR ORDERS yTCR Kit for the analysis of clonality of V J regions of the y chain of T cell receptor TCR cod 04 60A The kit contains the reagents for DNA amplification for visualization by agarose gel electrophoresis and positive control Target region T cell receptor gene TCR Cod Prod Pkg 04 60A 25 yTCR 25 tests 03 60A 50 yTCR 50 tests BanA
9. case the volume of sterile water in the reaction mix can be diminished Centrifuge briefly then incubate the tubes in the termalcycler programmed as below 1 cycle 94 C 5 min 94 C 30 sec 25 cycles 55 C 45 sec 73 C 45 sec 1 cycle 73 C 5 min storage 4 C Proceed directly with the ssDNA amplification Consumo pag 17 ven 04 60R 50 8033622780731 EN doc 11 2 2 TCR y CHAIN V J REGION ssDNA AMPLIFICATION Add to each green MM2 test tube AB Taq Put the obtained mix 21 uL in the MM1 tubes over the wax stratum paying attention not to damage it Centrifuge shortly and put the test tubes in the thermalcycler programmed as below 1 cycle 94 C 5 min 94 C 30 sec 30 cycles 55 C 45 sec 73 C 45 sec 1 cycle 73 C 7 min storage 4 C Amplification fragments length is in the range from 160 bp to 190 bp Danautice pag 18 04 60R 50 8033622780731 EN doc 11 3 VISUALIZATION OF THE AMPLIFICATION PRODUCTS 11 3 1 Agarose gel electrophoresis Preparation of a 3 agarose gel Weight 1 5 g of HR Agarose and pour it into 50 mL of 1X TAE Leave the solution on a magnetic stirring heater or in a microwave until the solution becomes clear Allow the gel to cool to hand warm and then add 10 uL of Ethidium Bromide solution NOTICE Ethidium Bromide is a strong mutagenic agent always wear gloves and preferably work under a chemical safety cabinet during th
10. cells tumours are related to different development phases While B cells tumours are referred to the entire phase of B cells development T cells tumours correspond to the precocious and latest stadiums of it the lack of involvement of the intermediate stadiums of development could be due to the fact that immature T cells if not rescued in a short time after the successive positive maturation are programmed for cellular death In these circumstances there is not sufficient time for tymocites to accumulate enough mutations for malignant transformation Zucchini A et al 2002 The availability of molecular techniques has allowed not only the increase of the capacity to diagnose the diseases but also the possibility of a correct classification of them Cossman et al 1998 The most important application of molecular methods in lymphoid diseases evaluation is the determination of cell clonality These techniques are considered as the gold standard for the clonality determination and are used when this information can not be obtained by the immuno pathologic techniques mostly for lymphoid diseases of T origin The molecular method allows to evaluate if the lymphoid abnormality is due to a neoplastic process therefore to a monoclonal proliferation or to a reactive benignant process to which polyclonal proliferation is associated BaNALITICA pag 9 04 60R 50 8033622780731 EN doc Since a neoplasy is the result of an unlimited gr
11. concentrated Ethidium Bromide use a chemical dispensing fume cabinet Always wear disposable gloves and laboratory coat in manipulating the diluted Ethidium solution as well The product can not be disposed with the common waste It must not reach the drainer system For the disposal follow the local law In case of accidental spilling of Ethidium Bromide clean with Sodium hypochloride and water Safety data sheet MSDS of Ethidium Bromide is available upon request BanALiTIcA ea pag 6 04 60R 50 8033622780731 EN doc 5 MATERIALS REQUIRED BUT NOT PROVIDED 5 1 Reagents Sterile DNase and RNase free water Distilled water 5 2 Instruments Laminar flow cabinet use is recommended while adding TAQ polvmerase to the amplification premix to avoid contamination it would be recommended to use another laminar flow cabinet to add the extracted DNA Micropipettes range 0 2 2 uL 0 5 10 pL 2 20 pL Thermalcycler Microcentrifuge max 12 000 14 000 rpm Balance Magnetic heating stirrer or microwave Chemical cabinet use is recommended in handling Ethidium Bromide Horizontal electrophoresis chamber for agarose minigel Power supply 50 150 V UV Transilluminator Photo camera or image analyzer 5 3 Materials Disposable gloves Disposable sterile filter tips range 0 2 2 uL 0 5 10 uL 2 20 uL Graduate cilinders 1 L for of TAE dilution Pyrex bottle or Becker for agarose gel preparation Parafilm
12. e handling of this reagent or gels containing it Place the gel into the appropriate gel casting tray with the comb placed in and allow the gel to cool at room temperature or in a fridge until the gel becomes solid When the gel is solidified remove carefully the comb pay attention not to damage the gel wells transfer the tray into the electrophoresis chamber and pour the appropriate amount of TAE buffer so that it covers completely the gel about 1 2 mm over the gel surface 11 3 2 Sample loading Mix into a tube or directly on a parafilm layer 4 uL of 6X Blue 20 uL of y TCR V J region amplification product OR 2 uL of 6X Blue 10 uL of DNA molecular weight marker MW Marker Load the mixture on the gel wells switch on the power supply and set the voltage at 50 V Run the gel for about 5 6 hours then place the gel on an UV transilluminator and analyze the results by comparing the size of the amplification products with the reference Molecular Weight Marker BanALITICA pag 19 04 60R 50 8033622780731 EN doc DNA Molecular Weight Marker Marker MW fragment sizes 501 489 404 353 242 190 147 110 89 67 34 26 bp NOTE In a 396 agarose gel the 501 489 bp bands are usually not clearly resolved and appear as an unique band the 26 and 34 bp bands are sometimes too small to be visible in a 3 agarose gel because of their low molecular weight NOTICE UV rays are dangerous for skin and above all ey
13. es always wear gloves and safety glass or make use of the protection screen of the UV transilluminator BANALITICA pag 20 04 60R 50 8033622780731 EN doc 11 4 INTERPRETATION OF THE RESULTS In order to discriminate a polyclonal sample from a monoclonal sample it is necessary to evaluate the clearness and the resolution of the bands of the amplification products This is possible only if the agarose gel preparation is accurate and if the electrophoretic run lasted for at least 5 hours For these reasons the correct amplification and visualisation of the monoclonal positive control included in the kit and of the negative polyclonal control any genomic DNA of a healthy patient are essential After agarose gel electrophoresis the controls should show the following results CONTROL RESULT INTERPRETATION Negative Control Shean Absence of contaminations water during the first amplification Monoclonal Wel dance vand Amplification and visualisation in the range 160 bp 190 bp Not well defined Polyclonal band or smears Amplification and visualisation negative control in the range were correct 160 bp 190 bp positive Control were correct Then for the interpretation of the band pattern follow the table below RESULT INTERPRETATION Presence of two well Presence of T cells MONO or defined bands BI CLONAL populations in the range 160 bp 190 bp Presence of a smear Presence of T cells POLYC
14. hniques applied to diagnostics e Before starting the kit procedure read carefully and completely the instruction manual e Keep the product out of heating sources e Do not use any part of the kit if over the expiration date e n 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 investigator has to take the following special precautions e Use filter tips e Store the biological samples the extracted DNA positive control included in the kit and all the amplification products in different places from where amplification reagents are stored Consumo pag 4 04 60R 50 8033622780731 EN doc e Organise the space in different pre and post PCR units do not share consumables pipets tips tubes etc between them e Change the gloves frequently e Wash the bench surfaces with 596 sodium hypochloride e Thaw the PCR premixes at room temperature before use Add the Taq DNA polymerase and purified DNA very quickly at room temperature or in an ice bath 4 SAFETY RULES 4 1 General safety 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 with 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 p
15. in most of neoplastic and normal T lymphocytes The TCR gene rearrangement takes place in a precocious stadium of T differentiation and particularly y 6 rearrangement precedes that of a f chains The ssDNA single strand DNA synthesis followed by y chain V J regions amplification guarantees to the method the necessary sensitivity for its application in the T cells monoclonality study The revelation of the amplified fragment is performed by a high resolution agarose gel electrophoresis The different aspects of the amplified products allow to discriminate a monoclonal sample from an oligoclonal or polyclonal sample The kit also provides with positive control of amplification When the amplification of the positive control is successful it is guaranteed that the reaction is correct These controls are not dangerous for the operator because they are plasmid DNA containing only the V J region of the y chain obtained from monoclonal T cells The kit is in premix format all the reagents for the amplification are pre mixed and aliquoted in monodose test tubes 0 2 or 0 5 mL to which Taq polymerase and the extracted DNA will be added This premix format allows the reduction of the manipulation steps in pre amplification with considerable time saving for the operator the repeated freezing thawing of reagents that could alter the product performances is avoided and above all this form minimizes the risk of contamination so the risk to get
16. otentially 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 Hypochloride 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 Hypochloride 1 volume of 5 Sodium Hypochloride 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 Hypochloride BanALITICA pag 5 04 60R 50 8033622780731 EN doc 4 2 Safetv rules about the kit The risks for the use of this kit are related to the single components Dangerous components 3 8 diamino 1 ethyl 6 phenylphenantridiumbromide Ethidium Bromide 296 Description of risk T Toxic 8 RISK SENTENCES AND S SENTENCES ETHIDIUM BROMIDE R23andR68 Toxic for inhalation Risk of irreversible effects S 36 37 45 Wear laboratory coat and disposable gloves In case of accident or discomfort seek for medical assistance and show the package label R and S sentences refer to the concentrated product as provided in the kit In particular for Ethidium Bromide until the dilution in the agarose gel In manipulating
17. owth of a single cell all the cells of a T cell cancer have the same rearranged TCR genes T Cell Receptor The TCR or T Cell Receptor is an hetherodimeric glycoprotein characterised by 4 distinct chains a f y and expressed as heterodimers a B or y The aminoacidic sequence analysis of the heterodimers revealed a surprising analogy between TCR structural dominium and those relating to the Immunoglobulin Ig this fact leads to consider the TCR as a member of the lg superfamily As for the lg genes the TCR variable dominium is produced as a result of the rearrangements of V variable and J Joining segments in the a and y families chains and of the V D diversity and J segments in the p and y families chains these regions are flanked by the constant C respective genic segments In particular the dominium nearest to the membrane is constant while the dominium far from the membrane is variable and contains the antigen binding site y chain rearrangement V genes J1genes Ctgenes J2genes C2genes VTH VII V3H Vn IHIHIHE MEN TID germline DNA rearrangement ea 4 V1 H V2H v3 J3 c2 l BANALITICA pag 10 04 60R 50 8033622780731 EN doc Most of T lymphocytes expresses the a B heterodimers the T lymphocytes percentage with o p or y varies in the different organs and from specie to specie in man the most of mature T cells located in the pe
18. ripheral blood expresses the a p TCR while T cells expressing y 6 TCR are in a little amount from 1 96 to 5 96 The complete and functional TCR is joined to CD3 a signal transduction molecular complex This structure is involved in the antigenic acknowledgment only after the antigen has been processed by an antigen presenting cell APC Before being recognised by T lymphocytes the antigen has to be fagocitated or endocytated by the APC and processed in little peptides Antigen fragments are then presented to TCR in the context of the products of and Ill class Major Histocompatibility Complex MHC that are constitutively expressed on the accessory cells membrane The contact between TCR and its specific antigenic determinant leads to the internalisation of the CD3 molecule The analysis of the TCR gene rearrangements by molecular biology techniques allow to detect the neoplastic nature of T cells expansions It is not possible instead to demonstrate the clonal nature of NK cells leukaemia whose diagnosis is done only on the basis of morphological immuno phenotypic and clinical analysis BanALITICA pag 11 04 60R 50 8033622780731 EN doc 8 TEST PRINCIPLE PCR method Polymerase Chain Reaction has been 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 polymera
19. se Three nucleic acid segments are involved in the reaction double stranded DNA template to be amplified target DNA and two single stranded oligonucleotides primers that are designed in order to anneal specifically to the template DNA The DNA polymerase begins the synthesis process at the region marked by the primers and synthesizes new double stranded DNA molecules identical to the original double stranded target DNA region by facilitating the binding and joining of the complementary nucleotides that are free in solution dNTPs After several cycles one can get millions of DNA molecules which correspond to the target sequence The sensitivity of this test makes it particularly suitable for the application in laboratory diagnostics Moreover the amplification reaction can be executed from a wide range of biological samples and since it allows to amplify very small DNA fragments the starting DNA can be also partially degraded BANALITICA pag 12 04 60R 50 8033622780731 EN doc 9 PRODUCT DESCRIPTION The molecular strategy adopted in this kit for detecting T cells monoclonality is characterised by the use of consensus primers for the V and J regions of the TCR y chain TCR y chain analysis is the simplest and most efficient method to study TCR gene rearrangements In effect with reference to p chain the number of gene segments of y chain coding locus is reduced and moreover y genes rearrangements are present
20. ture changes too slowly Do a thermalcycler revision e The preparation of the amplification reaction has been executed in a long time at room temperature Accelerate the work time at room temperature Work on ice e The extracted has not been purified Use an extraction system which allows a good sample purification anautica pag 23 04 60R 50 8033622780731 EN doc e The starting sample contained degraded DNA Repeat the extraction step using another clinical starting sample Be sure that the sample had been collected and stored in appropriate Way 3 The positive monoclonal control doesn t appear as a clear and well resolved band e The gel overheated decrease the voltage applied e The gel was not correctly prepared be sure to completely melt the agarose powder by mixing it with TBE 1X For any further problem contact AB ANALITICA technical support e mail laboratorio abanalitica it BANALITICA pag 24 04 60R 50 8033622780731 EN doc 13 DEVICE LIMITS The kit can have reduced performances if the blood sample is not suitable for this analysis not correct sample storing using of heparin as anticoagulant the histological sample is not suitable for this analysis using of alternative fixatives instead of neutral formalin buffer not correct sample storing 14 DEVICE PERFORMANCES 14 1 Specificity Primer sequence alignment in the most important data
21. ucocytes and the remaining fraction contains the erythrocytes Alternatively it is also possible to perform a FICOLL protocol in order to isolate a pellet of white cells 10 2 Histological samples Histological samples include fresh frozen or formalin fixed and paraffin embedded biopsies The fresh biopsv could be treated within few minutes from sampling or quickly frozen with liquid nitrogen and successively stored at 80 C until mechanic disruption by using sterile cutter followed by enzymatic digestion with Proteinase K In case that the biopsy is fixed and paraffin embedded it is suggested the use of formalin buffer at pH 7 with sodium and potassium salts at 1096 as Lilie formula Tissue fixation with not buffered formalin in Bouin Holland or other acidic fixatives osmic acid for instance is not suitable for subsequent DNA extraction because those substances produce cross links in the tissue making it not digestible Consumo man pag 14 04 60R 50 8033622780731 EN doc 10 2 1 Pre treatment of histological samples In case of fresh or frozen histological sample up to 50 mg proceed quickly with mechanic disruption of the sample by a sterile cutter Do this operation on a glass slide transfer the minced tissue in a tube then proceed with digestion with Proteinase K In case that the biopsy is fixed and paraffin embedded proceed first with paraffin removal and then with sample digestion BanALITICA pag 15
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