HLA B5701 Real
Contents
1. 2 D HEE GENTAUR ABATRE For in Vitro Diagnostic Use For Professional Use Only HLA B 5701 Real TM Handbook Real Time PCR test for the detection of HLA B major histocompatibility complex class I B Allele 5701 REF H53 100FRT YY 100 NAME HLA B 5701 Real TM INTRODUCTION Abacavir is a nucleoside reverse transcriptase inhibitor with activity against the human immunodeficiency virus HIV available for once daily use in combination with other antiretroviral agents that has shown efficacy few drug interactions and a favorable long term toxicity profile The most important adverse effect of abacavir that limits its use in therapy and mandates a high degree of clinical vigilance is an immunologically mediated hypersensitivity reaction affecting 5 to 8 of patients during the first 6 weeks of treatment Symptoms of a hypersensitivity reaction to abacavir are nonspecific and include combinations of fever rash constitutional symptoms gastrointestinal tract symptoms and respiratory symptoms that become more severe with continued dosing Immediate and permanent discontinuation of abacavir is mandated resulting in a rapid reversal of symptoms Subsequent rechallenge with abacavir is contraindicated since it can result in a more severe rapid and potentially life threatening reaction In 2002 an association between a diagnosis of hypersensitivity reaction to abacavir and carriage of the major histocompatibility complex clas2. be used for materials that contain or are suspected of containing infectious agents Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Avoid contact of specimens and reagents with the skin eyes and mucous membranes If these solutions come into contact rinse immediately with water and seek medical advice immediately Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of DNA amplification PCR reactions are sensitive to contamination Measures to reduce the risk of contamination in the laboratory include physically separating the activities involved in performing PCR in compliance with good laboratory practice Workflow in the laboratory must proceed in a uni directional manner beginning in the Extraction Area and moving to the Amplification and Detection Area Do not return samples equipment and reagents in the area where you performed previous step Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer Sampling of biological materials for PCR analysis transportation and storage are al described in details in the handbook of the manufacturer It is recommended that this handbook is read before beginning of the work SAMPLE COLLECTION STORAGE AND TRANSPORT HLA B 5701 Real TM can analyze genomic DNA
3. extracted from whole blood Collect 2 ml of blood to a tube with 0 2 ml of 3 EDTA solution Invert a closed tube several times to ensure proper mixing Blood samples should be stored at 2 8 C for up to 48 h Oropharyngeal swabs are taken with a sterile probe with a cotton tip After swabbing the probe should be placed to a tube with 0 5 ml of Transport Medium for Storage and Transportation Respiratory Swabs REF 958 The probe should be broken off at the score mark so that the tube is tightly closed The sample should be stored at 2 8 C for up to 3 days Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DNA ISOLATION The following isolation kits are recommended e DNA RNA Prep Sacace REF K 2 9 gt i samples Transportation of Respiratory Swabs REAGENT PREPARATION 1 Prepare the reaction mixture Per one reaction 10 pl of PCR mix 1 FRT HLA S5plof RT PCR mix 2 FL 0 5 pl of polymerase TaqF Add one extra reaction when calculating the reaction mixture volume Extract DNA according to the manufacturer s instruction Whole blood samples should be treated with Hemolytic REF 137 CE before adding the lysis solution To do this add 1 0 ml of Hemolytic and 0 1 ml of whole blood to a 1 5 ml tube Carefully vortex Incubate the tubes at room temperature for 5 min vortex and in
4. screening for hypersensitivity to abacavir Mallal et al N Engl J Med 2008 Feb 7 358 6 568 79 Value of the HLA B 5701 allele to predict abacavir hypersensitivity in Spaniards Rodr guez N voa S Garc a Gasc P Blanco F Gonz lez Pardo G Castellares C Moreno V Jim nez N cher I Gonz lez Lahoz J Soriano V AIDS Res Hum Retroviruses 2007 Nov 23 11 1374 6 Prospective HLA B 5701 screening and abacavir hypersensitivity a single centre experience Waters LJ Mandalia S Gazzard B Nelson M AIDS 2007 Nov 30 21 18 2533 4 Abacavir hypersensitivity reaction in primary HIV infection Stekler J Maenza J Stevens C Holte S Malhotra U McElrath MJ Corey L Collier AC AIDS 2006 Jun 12 20 9 1269 74 The pharmacogenetics of antiretroviral therapy Phillips EJ Curr Opin HIV AIDS 2006 May 1 3 249 56 iCycler and iQ5 are trademarks of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABI is a registered trademark of Applied Biosystems SmartCycler is a registered trademark of Cepheid Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium
5. cubate for 5 min once again Centrifuge 8 000 rpm 2 min Remove and discard the supernatant Leukocyte sediment should be immediately lysed otherwise it should be stored frozen at or below minus 16 C for up to 3 days or at or below minus 68 C for a long time Prior to DNA extraction from throat swabs placed in Transport Medium for Storage and 957 CE thoroughly mix and then briefly vortex the Volume of the reagents for specified number of samples ul Number of oe samples one extra reaction 1s included PCR mix 1 FRT HLA RT PCR mix 2 FL Polymerase TaqF 6 70 35 3 5 11 120 60 6 0 18 190 95 9 5 N Thoroughly vortex prepared mixture make sure there are no drops on the wall of the tubes 3 Take the required number of the PCR tubes for amplification of clinical and control samples Transfer 15 ul of prepared reaction mix to each tube 4 Add 10 ul of DNA samples obtained from clinical or control samples at the stage of DNA extraction into prepared tubes 5 Carry out control amplification reactions NCA Add 10 wl of TE buffer to the tube labeled NCA Negative Control of Amplification C Add 10 ul of Positive Control DNA HLA B 5701 and human DNA to the tube labeled C Positive Control of Amplification Create a temperature profile on your Real time instrument as follows Rotor type instruments Plate type or modular instruments Stage T
6. e that you use a recommended DNA extraction method and follow the manufacturer s instructions e The reagents storage conditions didn t comply with the instructions Check the storage conditions e The PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol e No correct sample collection or preparation 2 No signal on the Joe Yellow Cy3 HEX and Fam Green channels with Positive Control e The reagents storage conditions didn t comply with the instructions Check the storage conditions e The PCR conditions didn t comply with the instructions gt Check the temperature profile and select the fluorescence channel reported in the protocol e Incorrect configuration of the PCR reaction Check the reagents preparation step 3 Any signal with Negative Control e Contamination during PCR preparation procedure All samples results are invalid gt Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive controls at the end gt Repeat the PCR preparation with the new set of reagents 4 Variation of more than 5 cycles between Ct values of Fam Green and Joe Yellow in a sample e Contamination with genomic material during PCR preparation procedure Sample result is invalid retesting of sample is required PERFORMANCE CHARACTERISTICS Sensit
7. emp C Time HIMOTNCENCe Ode Temp C Time Fluorescence detection Cae detection repeats repeats Hold 95 15 min 1 95 15 min 1 95 5s 95 5s Cycling 5 5 60 20s 60 20 s 95 5s 95 5s Cycling 2 FAM Green 40 50 40 s 60 40s JOE Yellow 60 FAM JOE HEX Cy3 For example Rotor Gene 6000 Q Qiagen For example iQ5 iQ iCycler BioRad Mx3005P Agilent ABI 7300 7500 Real Time PCR Applied SmartCycler Cepheid RESULTS ANALYSIS The results are interpreted by the device software through the presence of crossing of fluorescence curve with the threshold line DNA HLA B5701 is detected on the JOE Yellow HEX Cy3 channel and JC on the FAM Green channel Results are accepted as relevant if both positive and negative controls of amplification along with negative control of extraction are passed see table 1 Table 1 Results for controls Control Stage lor Baa Interpretation control FAM Green JOE Y ellow HEX C DNA extraction Neg Neg OK NCA Amplification Neg Neg OK C Amplification Pos lt boundary Pos lt boundary OK value value e The sample is considered to be positive if in the channel Joe Yellow HEX Cy3 the result is positive and the value of Ct on this channel is higher than Ct on the Fam Green channel but not more than 5 cycles see table 2 e The sample is considered to be negative if in the channel Joe Yellow HEX Cy3 va
8. he presence of cellular material in the sample PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics Use of this product should be limited to personnel trained in the techniques of DNA amplification EN375 Strict compliance with the user manual is required for optimal PCR results Attention should be paid to expiration dates printed on the box and labels of all components Do not use a kit after its expiration date QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality MATERIALS PROVIDED Reagent Description Volume ml Amount PCR mix 1 FRT HLA colorless clear liquid 0 6 2 tubes RT PCR mix 2 FL colorless clear liquid 0 3 2 tubes Polymerase TaqF colorless clear liquid 0 03 2 tubes TE buffer colorless clear liquid 0 07 2 tubes court co ec Bae band colorless clear liquid 0 2 1 tube Negative Control C colorless clear liquid 0 5 4 tubes must be used in the isolation procedure as Negative Control of Extraction MATERIALS REQUIRED BUT NOT PROVIDED DNA extraction kit Disposable powder free gloves Pipettes adjustable Sterile pipette tips with aerosol filters up to 200 ul Tube racks Vortex mixer Desktop centrifuge with a rotor for 2 ml reaction tubes PCR box Personal thermocycler for example Rotor Gene 6000 Q Qiagen
9. iQ5 Bio Rad USA or equivalent Disposable polypropylene microtubes for PCR or PCR plate Refrigerator for 2 8 C Deep freezer for lt 16 C Waste bin for used tips STORAGE INSTRUCTIONS The kit HLA B 5701 Real TM must be stored at or below minus 16 C when not in use The kit can be shipped at 2 8 C for 3 4 days but should be stored at 20 C immediately on receipt A N PCR mix 1 FRT HLA is to be kept away from light STABILITY HLA B 5701 Real TM is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label The shelf life of reagents before and after the first use is the same unless otherwise stated Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should be avoided as this may reduce the sensitivity WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device 1 Sy PD 11 12 For In Vitro Diagnostic Use Only Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward Do not pipette by mouth Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Dispose of all specimens and unused reagents in accordance with local regulations Biosafety Level 2 should
10. ivity Analytical Sensitivity of HLA B 5701 Real TM PCR kit is not less than 1 x 10 cells per 1 ml of a sample cells ml The claimed analytical features of HLA B 5701 Real TM PCR kit are guaranteed only when additional reagents kit DNA RNA Prep Sacace REF K 2 9 is used Specificity Specificity of HLA B 5701 Real TM PCR kit is assured by selection of specific primers and probes as well as the selection of strict reaction conditions The primers and probes were checked for possible homologies to all in gene banks published sequences by sequence comparison analysis Specificity of HLA B 5701 Real TM PCR kit was confirmed in laboratory clinical trials KEY TO SYMBOLS USED LOT References List Number Lot Number Caution Contains sufficient for lt n gt tests fs in Vitro Diagnostic VER verion se Negative Control of Store at NCA Amplification Negative control of ual Manufacturer cC Extradiiot Ci Consult instructions for c Positive Control of use Amplification Expiration Date IC Internal Control High sensitivity of human leukocyte antigen B 5701 as a marker of immunologically confirmed abacavir hypersensitivity in white and black patients Saag M et al Clin Infect Dis 46 1111 1118 2008 Association between presence of HLA B 5701 HLA DR7 and HLA DQ3 and hypersensitivity to HIV 1 reverse transcriptase inhibitor abacavir Mallal S Nolan D et al Lancet 2002 Mar 2 359 9308 722 3 HLA B 5701
11. lue is negative or if the value of Ct on this channel is higher than Ct on the Fam Green of more than 5 cycles e Normal difference between Joe Yellow and Fam Green Ct values is 2 3 cycles Table 1 Results for samples Ct value and result Sample RotorGene iQ iQ5 Mx3005 ABI FAM IC JOE HLA FAM IC HEX HLA C lt 25 lt 25 lt 29 lt 29 positive positive positive positive Clinical T lt 25 lt Ct FAM 5 lt 29 lt Ct FAM 5 positive positive positive positive QUALITY CONTROL PROCEDURE HLA B 5701 Real TM PCR kit is a qualitative test which contains the Internal Control IC human beta globine gene which allows to control the presence of cellular material in the sample If the sample is not correctly prepared or it is an insufficient quantity of epithelial cells the Internal Control will not be detected A negative control of extraction NCE negative amplification control NCA positive amplification control C are required for every run to verify that the specimen preparation the amplification and the detection steps are performed correctly If the controls are out of their expected range see table Results for Controls all of the specimens and controls from that run must be processed beginning from the sample preparation step TROUBLESHOOTING 1 Absent signal of the IC Fam Green channel retesting of the sample is required e The PCR was inhibited Make sur
12. s I allelee HLA B 5701 was reported independently by several independent studies Studies of cohorts with HIV infection have also shown that avoiding abacavir in HLA B 5701 positive patients significantly reduced the incidence of suspected hypersensitivity reaction up to 0 5 Many clinical studies recommend for this reason the pharmacogenetic molecular testing of the carriage of the major histocompatibility complex class I allelee HLA B 5701 in all HIV positive patients treated with abacovir HLA B 5701 Real TM test can predict who will develop a severe allergic reaction to the anti HIV drug abacavir as the presence of HLA B 5701 is significantly associated with an abacavir hypersensitivity INTENDED USE HLA B 5701 Real TM is a Real Time amplification test for the detection of HLA B major histocompatibility complex class I B Allele 5701 in the biological materials The kit HLA B 5701 Real TM can be used as screening test for the prevention of abacavir hypersensitivity reactions PRINCIPLE OF ASSAY HLA B 5701 Real TM Test is based on two major processes isolation of genomic DNA from specimens and Real Time amplification with allele specific primers The real time PCR monitoring of fluorescence intensities allows the accumulating product detection without reopening of reaction tubes after the PCR run HLA B 5701 Real TM PCR kit is a qualitative test which contains the Internal Control IC human beta globine gene which allows to control t
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