How To Set Up A Molecular Pathology Lab: A Guide For Pathologists

1m ago
739.11 KB
9 Pages
Last View : 1m ago
Last Download : n/a
Upload by : Warren Adams

Reviewdoi: 10.5146/tjpath.2020.01488How to Set Up a Molecular Pathology Lab:A Guide for PathologistsAnıl AYSAL, Burçin PEHLIVANOĞLU, Sümeyye EKMEKCI, Betül GÜNDOĞDUDepartment of Molecular Pathology, Dokuz Eylul University, Graduate School of Health Sciences, IZMIR, TURKEYABSTRACTIn today’s pathology practice, pathologists combine molecular tests with conventional histopathological methods. Pathology laboratoriesshould therefore be designed and operated in accordance with the requirements of molecular testing procedures. While the specifics of therequirements may vary depending on the spectrum of the tests that will be performed, there are several basic criteria that need to be fulfilledfor standardization. Adequate space, appropriate equipment and qualified personnel are required to establish a molecular pathology laboratory.One of the most important points that should be taken into consideration while designing a molecular pathology laboratory is to create a plan toprevent contamination. As molecular diagnosis has a major role in treatment decisions, the management of the molecular pathology laboratoryis of utmost importance. In this review, the criteria required to establish an optimal molecular pathology laboratory will be reviewed.Key Words: Molecular pathology laboratory, Laboratory setup, GuideINTRODUCTIONThe effects of diseases on the human body were firstdocumented by ancient Egyptians however, the concept oforgan-specific disease and anatomic pathology have begunto evolve only in the last few centuries, and the alterationsat the tissue and cellular level have gained attentionfollowing the invention of the microscope (1). Themolecular pathology era has begun with the integrationof molecular tests into pathology practice, especially indiagnosis of solid tumors and hematological malignancies,as a part of the improvements in molecular sciences thathad been led by the completion of the Human GenomeProject in early 2000s. Pathologists now have a critical roleof morphomolecular assessment in this new generationmedicine era (2) and therefore have critical impact on thepreanalytical phase of molecular testing (3). Given thatpathologists combine molecular tests with conventionalpathological evaluation methods, pathology laboratoriesshould be designed and operated in accordance with therequirements of molecular testing procedures.Adequate space, appropriate equipment and qualifiedpersonnel are required to establish a molecular pathologylaboratory. While the specifics of the requirements mayvary depending on the spectrum of the tests that will beperformed, there are several basic criteria that need tobe fulfilled for standardization. In this paper, the criteriarequired to establish a molecular pathology laboratory willbe reviewed.(Turk Patoloji Derg 2020, 36:179-187)Received : 28.01.2020 Accepted : 09.04.2020Required Physical ConditionsOne of the most important points that should be takeninto consideration while designing a molecular pathologylaboratory is to create a plan to prevent contamination.Polymerase chain reaction (PCR)-based methods areespecially susceptible to contamination (4). To obtaina large number of copies from a very small amount ofthe target sequence with the PCR method provides animportant diagnostic advantage, but this ability also leads tofalse results in case of contamination. False positive resultsmay occur due to contamination from sample to sample,transport of amplicons from the previous amplificationof the same target, cross-contamination of differentreactions prepared simultaneously, and contaminationof the reagents with DNA templates (5). In real-timePCR methods; when the PCR reactions are finished withfluorescence-based detection techniques, the analysis isalso completed at the same time. Since the PCR productsdo not need to be reprocessed, the reaction tubes or closedplates are not opened and the amplicon transport does notoccur (4). The laboratories using real-time PCR methodstherefore have less risk of contamination (5).Main procedures performed in a molecular pathologylaboratory using PCR-based methods are pre-PCRprocedures (sample preparation, PCR preparation) andpost-PCR procedures (performing PCR and post-PCRanalysis) (4). It is critical to perform these operations inareas separated as “clean” and “dirty”. The “clean” areaCorrespondence: Anıl AYSALDokuz Eylul University, Graduate School of Health Sciences,Department of Molecular Pathology, IZMIR, TURKEYE-mail: Phone: 90 232 412 34 40179

Turkish Journal of PathologyAYSAL A et al: How to Set Up a Molecular Pathology Labrepresents the area where all pre-PCR procedures (such asmicrodissection, DNA/RNA extraction, PCR preparation)are performed, and the “dirty” area represents the areawhere all post-PCR products (amplicons) are processed.The staff and researchers should keep all reagents,materials and equipment used in these areas separate atall times and never move them back from the dirty area tothe clean area (6). Contamination is significantly reducedby physical separation of the clean and dirty areas and bydoing pre-PCR and post-PCR activities in separate rooms.Therefore, planning at least two separate rooms is essentialwhile designing a molecular pathology laboratory. It isrecommended to perform sample preparation steps suchas nucleic acid isolation in the pre-PCR laboratory, and toperform PCR reactions and other post-PCR procedures inthe post-PCR laboratory (4). However, if there is sufficientspace, four separate rooms are recommended for thepreparation of reagents, sample preparation, PCR stepand post-PCR steps for an ideal molecular pathologylaboratory. Each room must have its own equipment,protective clothing and consumables, and there should beno material/equipment transport between the rooms (7).The requirements for laboratory design may vary accordingto the method used. For example, as mentioned previously,3 rooms may be ideal in a laboratory where a real-time PCRmethod is applied, since post-PCR analysis is not necessaryin the real-time PCR method (5).rooms (5). These rooms are also called “high copy” rooms(8). In the PCR applications such as real-time PCR wheresingle-stage PCR reactions are sufficient and tubes withPCR products are not required to be opened, PCR devicescan be placed in the post-PCR room. However, in thelaboratories using PCR applications such as nested PCR,etc., where multiple PCR reaction steps are required andthe tubes must be opened, PCR devices should be placedin a separate room/area (9). In the amplification phase,the primary and secondary PCR steps (if any), should beseparated according to the physical state of the laboratory,preferably in separate rooms. If this is not possible, theyshould be performed in separate compartments and onseparate PCR devices (7). Next-generation sequencingapplications also include one or more PCR amplificationsteps, which are similarly recommended to be performedin separate rooms/areas (5). Various recommendationsabout minimum room sizes can be found in internationalguidelines. For example, according to the field planningcriteria of the United States of America Military HealthSystem Pathology and Clinical Laboratories guide, thereagent preparation room should be at least 120 sq ft(approx. 33.5 m2) and the amplification room should be240 sq ft (approx. 22.3 m2) in size (10). The relevant guidepublished by the Republic of Turkey Ministry of Health isdetailed below.Reagent preparation room is the room where reagentstocks are prepared and then divided into a certain numberof small usable parts (aliquoted), and the reaction mixesare prepared. This room should be free of any biologicalmaterials such as DNA/RNA extracts, PCR products, etc.The sample separation room is where the nucleic acidisolation is performed and the isolated samples are addedto the PCR reaction mixes (5). This room is also called a“low copy” room, as the number of copies has not yet beenamplified by PCR (8). Ideally, it is recommended to performthe steps of nucleic acid isolation and addition of isolatedsamples to the PCR reaction mixes in separate rooms, butthese two steps are usually performed in the same room butin different areas/compartments since most laboratoriesdo not have sufficient space (5). Preparation of the PCRreactions in a laminar flow biosafety cabinet ensures thatthe area remains clean (9). The amplification (PCR) roomis where PCR devices are located and the amplificationsteps are performed, and the post-PCR room is where theanalysis of PCR products by gel electrophoresis, sequencing,nested-PCR, etc., methods are carried out. These two roomsconstitute contaminated-dirty rooms and no equipmentor materials used in these rooms should be used in otherThe workflow in the molecular pathology laboratory mustbe unidirectional from the clean area to the dirty area (7).When laboratory personnel and researchers are required tomove from dirty rooms to clean rooms, laboratory coats,gloves and all kinds of protective equipment should bechanged and hands should be washed. No material shouldbe carried from the dirty room to the clean room (5). Toprevent the passage of personnel from the dirty room tothe clean room, it is appropriate to have separate personnelworking in each room or to perform pre-PCR and postPCR procedures on different days (9). There are automatedmolecular pathology platform systems that provideautomatic one-way workflow and isolate nucleic acid fromthe sample, combine the isolated DNA with amplificationreagents, and perform the analysis, and their use is becomingincreasingly common (5,11). The rooms and workflowthat should be present in an ideal molecular pathologylaboratory are shown in Figure 1. If all operations have tobe performed in a single room, separate compartments/benches are required for the reagent preparation, samplepreparation, PCR stage and post-PCR stages. The rule ofunidirectional workflow from the clean compartmentsto the dirty compartments must be followed. If possible,180WorkflowVol. 36, No. 3, 2020; Page 179-187

Turkish Journal of PathologyAYSAL A et al: How to Set Up a Molecular Pathology Labpressure, the air pressure inside the room is higher thanthe air pressure outside the room, preventing the transportof unwanted substances from outside. Negative pressure,on the other hand, allows air to enter into the room andprevents the migration of the air to the surrounding rooms/laboratories. The doors must be kept closed to maintainthe negative pressure. There should be slight positivepressure in the pre-PCR laboratory to prevent the entranceof contaminated air from outside, while the post-PCRlaboratory should have a slight negative pressure to keepthe air in and thus to prevent the escape of amplicons fromthe completed PCR samples. The ventilation of pre-PCRand post-PCR laboratories should be opened to differentair channels and opened out from different locations (4).Ultra-Violet (UV) IrradiationFigure 1: The Design and Workflow of an Ideal MolecularPathology Laboratory (Modified from references 4 and 8).sample preparation should be carried out in a laminar flowbiosafety cabinet including UV light. In the absence ofseparate rooms, a timetable should be established in whichthe pre-PCR and post-PCR steps are performed at differenttimes of the day (4).VentilationCirculating air between pre- and post-PCR laboratories isan important source of contamination in laboratories wheretechniques detecting very small amounts of DNA/RNA areused. Each laboratory should be ventilated separately andthe air pressure must be adjusted separately. At positiveVol. 36, No. 3, 2020; Page 179-187UV rays that cause DNA damage are useful for eliminatingthe contaminated DNA that may occur during additionof the DNA template. UV light can therefore be used tosterilize the pre-PCR laboratory. Since this method isbased on cross-linking with thymidine residues, the basesequence of the target region plays a role in its success. Inaddition, the hydration status of the DNA has a significanteffect on the UV resistance of the DNA. As dry-state DNAis more resistant to UV light, UV light is less effective inpreventing contamination on dry laboratory surfaces(9). If UV light is going to be used on master mixes fordecontamination, care must be taken to ensure that nodNTPs and enzymes are damaged in the UV light (8). TheUV light source can be placed on the laboratory ceiling orbench and can be activated by a device on the exit dooras the last person leaving the laboratory closes the outerdoor. If UV lights are used, UV-induced ozone must beremoved by ventilation. Accumulation of deposits dueto the precipitation of oxidation products on the glassof the bulb during radiation occurs and this reduces theeffectiveness of the UV system. These deposits should beremoved monthly and the performance of the UV bulbsmust be strictly monitored (4).The physical conditions required in molecular laboratoriesin Turkey are defined by the “Guideline for PhysicalInfrastructural Standards of Medical Laboratories applyingMolecular Tests” published by the Republic of TurkeyMinistry of Health (12). According to this guideline; Molecular diagnostic laboratories should have at leasttwo, preferably three rooms, each with a minimumarea of 15 square meters, physically separated fromeach other to allow unidirectional workflow (frompreamplification to postamplification) and preferablywith separate ventilation systems.181

Turkish Journal of Pathology These rooms are defined in parallel with thoserecommended in the literature and internationalguidelines, as a ‘pre-amplification laboratory’ wheresample acceptance and nucleic acid extraction areperformed, an ‘amplification laboratory’ where targetamplification methods are applied, and a ‘postamplification laboratory’ where analysis methods suchas electrophoresis, DNA sequence analysis etc. takeplace after amplification. In order to prevent contamination, the issues ofproviding a clean and preferably separate airflow,preparation and storage of all reagents and chemicals intheir own areas, use of separate devices and materials foreach laboratory area (freezers, refrigerators, cabinets,centrifuges, water baths, vortex mixers, pipettes, pencils,timers, all kinds of consumables, etc.) are pointed out. It is emphasized that if only two rooms can be reservedfor the molecular diagnosis laboratory, preamplificationprocedures and amplification / postamplificationanalyses should be performed in separate rooms. For each laboratory; the issues of the presence of singlepiece floor covering without pores, the presence ofhand washing sinks, the provision of temperatureand humidity monitoring, the use of UV irradiationsystems (on counter tops and / or room ceilings) duringnon-working hours, the presence of sufficient storagespace, the presence of a sufficient number of groundedelectrical outlets and uninterruptible power supplies(UPS, generator, etc.) for the laboratory devices,and the placement of laboratory equipment to allowunidirectional workflow are pointed out.What to Do to Avoid Intermixing and Contamination ofSamplesMolecular laboratory tests are generally quite sensitive andspecific, providing very precise results (6). Even so, falsepositive or false negative results may sometimes occur.Control mechanisms that include the verification of theprimer and probe sequences, checking and confirmingwhether the test conditions are optimal, and the use ofnegative controls (9) should be employed to reduce falseresults.While amplification is generally a part of the moleculardiagnostic method, current nucleic acid amplificationmethods are very sensitive with the capability of detectingeven a single molecule. Although this seems to be anadvantage, it should be kept in mind that the contaminatedDNA molecule may also be amplified, causing false positive182AYSAL A et al: How to Set Up a Molecular Pathology Labresults. Therefore, prevention of contamination must be apriority in molecular pathology laboratories (9).Cross-contamination is one of the sources of error andcontamination in the pathology laboratory and may occurat any stage of the tissue processing process, such as duringmacroscopic and/or microscopic evaluation, or duringDNA extraction, and may cause false positive or negativeresults. Microorganisms (viruses, bacteria, etc.) may also betransferred from one case to another during these processes.Immunohistochemical staining with ABH blood groupantibodies, microdissection, and microsatellite instabilityanalysis can be performed to prevent and detect crosscontamination (2,13). As processing of the samples fromdifferent patients occurs in the same area with recurrentuse of several instruments (e.g., microtome blade, waterbath) in pathology laboratories, precautions such as usinga new blade for each sample, washing the blade with DNAdecontamination solution, and/or sectioning of an emptyparaffin block between samples (called the “sandwichmodel”) are used by various laboratories to reduce the risk ofcross-contamination (13). However, cross-contaminationrates have been reported to be around 3% (0-8.8 %) despitethese precautions (14).The amplified DNA from the positive reactions in theprevious test, when the reaction tubes are opened afteramplification, is the source of contamination for thesubsequent tests (9). Also, amplification reactions areexposed to contamination from other patients’ samplesand the target-containing plasmid (15). Samples maycontaminate the laboratory environment while pipettingand the risk of contamination increases if multiple samplesare run together. Positive controls studied in the test arerisk sources for contamination as well. Clothing, laboratorywaste and/or uncleaned tables may contain contaminatingnucleic acids (9).In order to prevent and control contamination, theappropriate physical conditions, architectural structureand design, meticulous application of laboratorytechniques and environmental control protocols, andthe workflow plan are essential. Using separate areas orrooms for pre-amplification, amplification and postamplification stages with separate ventilation systems isan efficient way to prevent contamination, as discussedin detail in the “Physical Conditions” section above. Therisk of contamination is lower in closed system devices.Only personnel in charge should be present in the testarea. Every area/room should have its own equipmentincluding laboratory coats and pipettes (4,6,15-17).Vol. 36, No. 3, 2020; Page 179-187

Turkish Journal of PathologyAYSAL A et al: How to Set Up a Molecular Pathology LabMinimum aerosolization while opening tubes is alsonecessary to prevent transport between samples (15).solutions and tubes are among the laboratory precautionsto prevent RNAase contamination (19).Cleaning before and after each procedure should be carriedout by using nucleic acid removers. For example, washingwith 10% bleach that is freshly prepared and rinsing with70% ethanol can be performed (16). Thus, both biologicallyhazardous substances and nucleic acids that may besources of contamination can be removed (15). Addingenzymes such as uracil DNA glycosylase, which breakdown DNA, into the amplified DNA to exchange some orall of the thymidine with uracil in the reaction productscan prevent contamination biochemically (18). In addition,contamination can be prevented or reduced by discardingunopened tubes in the last stage, using pipettes withpositive pressure displacements, not talking during testssuch as PCR, and regular exposure of laboratory devicesto UV radiation (4,6). Aliquoting the reagents for each runis another precaution for the prevention of contamination.Patient samples and positive controls should be the last tobe put into reaction tubes to reduce the risk of transport ofthe nucleic acids. If positive controls are going to be used,the lowest dilutions should be preferred (15).Preparation of a documented action plan in case ofcontamination is recommended. Most laboratoriesquarantine and/or destroy all contaminated reagents andconsumables (15).Water or DNA-free buffers can be used as negative controlsto detect and monitor contamination. The control tubeshould contain all materials for all stages of the test, likeany other sample. A positive result detected in the negativecontrol indicates the possibility of contamination (4,9).The adequacy control of chemical sterilization (such asuracil glycosylase protocols) can be done by incorporatinga small amount of amplicon into a negative control (15).Surface and equipment contamination can be checked byswab samples from the laboratory surfaces where the test iscarried out with damp filter papers, and a positive result inthe swab sample indicates the presence of contamination.In addition, more than expected positivity rates of a giventest may indicate contamination (4,9).As RNA is more reactive than DNA and is vulnerable toRNAses that are present in all cells, prevention of RNAsecontamination is very important in RNA-based moleculartests. RNases are resistant to metal chelating agents and canpersist even after prolonged boiling or autoclaving. Themost common sources of exogenous RNAse contaminationare contaminated buffers and automatic pipettors. Also,all laboratory surfaces and glassware can be contaminatedwith RNAse from the laboratory personnel’s skin, hair, etc.Wearing gloves and changing them frequently during allstages of the test, use of separate laboratory equipment forRNA based tests, aliquoting small amounts of buffers, useof RNAse inhibitors (DEPC, etc.), and use of RNAase-freeVol. 36, No. 3, 2020; Page 179-187EquipmentEquipment and appliances may slightly differ betweenmolecular pathology laboratories based on their testingprofile. On the other hand, often a detailed inventorylist (Table I) is required to set up a molecular pathologylaboratory and provide standard testing. It should also benoted here that equipment and consumables used in theroutine pathology setting must be available in a molecularpathology laboratory, as well. It is also important to budgetfor service contracts for maintenance and repair (6).Unidirectional workflow should be taken into considerationwhile organizing/placing the equipment (Figure 2). The listof the appliances and equipment required in a molecularpathology laboratory provided by Republic of TurkeyMinistry of Health is also a useful guide (Table II) (12).Instructions for calibration and maintenance should bekept in the laboratory as written guidelines. Calibrationguidelines must include the schedule for calibration (e.g.,daily, monthly etc.), instructions describing the steps of thecalibration procedure, calibration material specifications,preparation and storage conditions, troubleshooting anddocumentation methods, maintenance guidelines, theschedule for maintenance, instructions for performingmaintenance, and troubleshooting guidelines (20).Quality AssessmentQuality management is essential in all steps of pathologyevaluation (i.e., pre-analytical, analytical and postanalytical) and a very important component in molecularpathology practice. However, as the details of qualitymanagement are beyond the scope of this review, only thebasic principles will be mentioned here.There are several regulatory guidelines, including standardoperating procedure manuals, to be followed to set-up and/or manage a molecular pathology laboratory (12,17,20).The presence of errors affecting the accuracy of the resultsin a molecular pathology laboratory should be checkedregularly under the supervision of the pathologist (“qualitycontrol; QC”) to prevent or minimize erroneous reportsand provide the confidence that quality requirements willbe fulfilled (“quality assurance; QA) (21).183

Turkish Journal of PathologyAYSAL A et al: How to Set Up a Molecular Pathology LabTable I: The list of equipment and appliances required in a molecular pathology laboratory.EquipmentPurpose of UseRefrigeratorStorage of chemical kits and PCR products-20 C and -80 C freezerStorage of tissue culture and frozen samplesVortex mixerMixing small vialsRefrigerated and non-refrigerated centrifuge Separation of components based on densityThermal cyclerPolymerase chain reactionNucleic acid quantification by measuring the amount of light absorbed bySpectrophotometera sampleSequencerSequencingMicroscope equipped with a camera system Evaluation and archiving of test results (e.g., in situ hybridization)HybridizerIn situ hybridizationNucleic acid quantification instrumentNucleic acid quantificationElectrophoresis systemAnalysis of PCR productsUltraviolet light illuminatorTo prevent contamination via ultraviolet light ning optimal conditions (e.g., temperature)Emergency power source to maintain power input when/in case mainUninterruptible power sourcepower source failsLiquid nitrogen tankSnap freezingMicrowaveHeating, drying, vaporizingpH meterpH measurementMicrotomeSectioningWater bathIncubating samples at a constant temperatureSlide ovenSlide drying, sterilization, maintaining constant/certain temperature levelsTissue processorTissue processingTissue embedding machineTissue embeddingComputer, private network and softwareBioinformaticsSlide stainerStainingCoverslipperTo cover slidesImmunohistochemistry staining systemImmunohistochemical stainingAutomated pipetting systemTransfer and handling of precise volumes of samples and reagentsBiosafety cabinetTo prevent contaminationTurn-around time and test result statistics should bechecked and validated by using standard validation studies(22). For internal quality assessment (IQA), the use ofcontrol materials is recommended (see previous sectionsfor details).In addition to the internal precautions mentioned in theprevious sections, external quality assessment (EQA) mustalso be performed at given intervals for specific types oftests, as it is the most critical stage of quality management.184EQA, a measure of laboratory performance (23), has beenshown to be helpful to improve molecular pathologylaboratories (24), and EQA programs are the key elementsof a laboratory’s QA framework (25). Regular participationin EQA is needed to verify and improve the quality oftesting, as molecular pathology EQA schemes score thereport and the test result (26,27). As a part of an EQAscheme, participants receive test samples and their resultsare then reviewed to check for errors.Vol. 36, No. 3, 2020; Page 179-187

AYSAL A et al: How to Set Up a Molecular Pathology LabTurkish Journal of PathologyTable II: The list of appliances and equipment required in a molecular laboratory as set by the Republic of TurkeyMinistry of Health (12).Required appliances and equipment§ -20 C freezer§ 4 C refrigerator§ Automated pipetting system (minimum two sets: 0,1-10 μl; 10-20 μl; 10-100 μl, 20-200 μl; 200-1000 μl)§ Class II biosafety cabinet§ PCR cabinet§ Dry heat block and/or water bath§ Spectrophotometer§ Thermal cycler§ Electrophoresis system§ Gel imaging system (ultraviolet light based or computed)§ Vortex mixer§ Refrigerated centrifuge (min. 5000 rpm)§ Microcentrifuge (min. 15000 rpm for 1.5 ml tubes)§ Magnetic stirrer§ Analytical balance§ pH meter§ Microwave§ Relevant plastic and glass instrumentsRecommended appliances and equipment§ -80 C freezer§ Ice machine§ DNA sequencer§ Water deionizer system§ Hybridizer§ Fume hoodFigure 2: Equipment required in a two-room PCR laboratory (Modified from reference no. 4); *: must be kept on different benches/ indifferent areas.Vol. 36, No. 3, 2020; Page 179-187185

Turkish Journal of PathologyThe reports are scored and the participant laboratoriesgain the opportunity to improve their service. Laboratoriesacross Europe are also required to have accreditation(26). Accreditation is a process in which an authorizedindependent body officially recognizes that the laboratoryis competent to perform certain tasks, and may beconsidered the most effective system for QA as compliancewith ISO standards is checked by accreditation bodies. Bothaccreditation and participation in EQA are recognized aseffective and important tools to improve the accuracy andreliability of molecular testing (28).CONCLUSIONIn conclusion, if the results obtained by molecular diagnostictests are inaccurate due to any of the factors mentionedhere, serious problems may arise which adversely affect thediagnosis and treatment decision. As molecular diagnosishas a major role in treatment decisions, especially for cancerpatients, the management of the molecular pathologylaboratory is of utmost importance.CONFLICT of INTERESTThe authors declare no conflict of interest.ACKNOWLEDGEMENTWe would like to thank to Professor Kutsal Yörükoğlu andProfessor Sülen Sarıoğlu for their guidance and valuablecontributions during the writing of this article.AYSAL A et al: How to Set Up a Molecular Pathology Lab6. Hunt JL. Molecular pathology in anatomic pathology practice: Areview of basic principles. Arch Pathol Lab Med. 2008;132:24860.7. McDonagh S. Equipping and Establishing a PCR Laboratory. In:2nd ed. Bartlett J, Stirling D, editors. PCR Protocols: Methods inMolecular Biology. New Jersey: Humana Press; 2003 (226). 15-20.8. Khan Z. Setup of a PCR Laboratory. In: Park DJ, editor. Methodsin Molecular Biology. New York: Springer Science BusinessMedia; 2011. 3-14.9. Lo YMD, Chan KCA. Setting Up a Polymerase Chain ReactionLaboratory. In: Lo YMD, Chiu RWK, Chan KCA, editors. ClinicalApplications of PCR. Methods in Molecular Biology. New Jersey:Humana Press; 2006 (336). 11-8.10. Defense Health Agency Facilities. Chapter 530: Pathology andClinical Laboratory, DoD Space Planning Criteria. (Accessed onJanuary 28 2020). Available from: healthfac 530 2017.pdf11. Felder RA, Jackson KD, Walter AM. Process evaluation of anopen architecture real-time molecular laboratory platform. J LabAutom. 2014;19:468-73.12. T.

pathologists combine molecular tests with conventional pathological evaluation methods, pathology laboratories should be designed and operated in accordance with the requirements of molecular testing procedures. Adequate space, appropriate equipment and qualified personnel are required to establish a molecular pathology laboratory.

Related Documents:

work/products (Beading, Candles, Carving, Food Products, Soap, Weaving, etc.) ⃝I understand that if my work contains Indigenous visual representation that it is a reflection of the Indigenous culture of my native region. ⃝To the best of my knowledge, my work/products fall within Craft Council standards and expectations with respect to

101 27 35 11 28 # TEAM TOTAL SET #1 SET #2 SET #3 SET #4 SET #5 SET #6 SET #7 SET #8 1 Fashingbauer 149 13 11 3 36 16 24 21 25 2 Thapa 394 53 82 55 49 63 33 49 10 # TEAM IMPs Total SET #1 SET #2 SET #3 SET #4 . He is a member of ACBL Unit 134, and he is currently an NABC Master. Reese is also the captain of his high school golf team.

akuntansi musyarakah (sak no 106) Ayat tentang Musyarakah (Q.S. 39; 29) لًََّز ãَ åِاَ óِ îَخظَْ ó Þَْ ë Þٍجُزَِ ß ا äًَّ àَط لًَّجُرَ íَ åَ îظُِ Ûاَش

Collectively make tawbah to Allāh S so that you may acquire falāḥ [of this world and the Hereafter]. (24:31) The one who repents also becomes the beloved of Allāh S, Âَْ Èِﺑاﻮَّﺘﻟاَّﺐُّ ßُِ çﻪَّٰﻠﻟانَّاِ Verily, Allāh S loves those who are most repenting. (2:22

5 Read to Me Books (continued) SKU Description ISBN Price Qty Total SB1278 Reach for the Stars 978-1-59577-127-8 4.95/ea CDSET02 Read-Along CD Set (4 CDs) 978-1-59577-273-2 9.95/set PKFO Set of 10 Folk Tales and Fables Books 978-1-59577-255-8 44.95/set PKFO-SET Set of 10 Folk Tales and Fables Books with Read-Along CDs 978-1-59577-258-9 49.95/set PKNF Set of 9 N

Set Cardinality The cardinality of a set is the number of distinct elements in the set The cardinality of a set A is denoted n (A ) or jA j If the cardinality of a set is a particular whole number, we call that set a nite set If a set

1981 Honda C70 Passport Parts Catalog Page 11 Carburetor Parts List Diagram No. Description Honda Part No. 1 GASKET SET 16010-086-701 2 NEEDLE SET, JET 16012-174-671 3 FLOAT SET 16013-883-005 4 TOP SET 16014-086-305 5 CHAMBER SET, FLOAT 16015-174-671 6 SCREW SET 16016-174-671 7 VALVE SET, THROTTLE 16022-173-004 8 CARBURETOR ASSY.

From Land to Sea stamp set (A card 1) Fresh Fruit stamp set (A card 1, B card 1, C card 1) Sprinkles of Life stamp set (A card 2) Balloon Builders stamp set (B card 1, C card 1) Thankful Thoughts stamp set (B card 2) No Bones About It stamp set (C cards 3 & 4, D card 2) Tin of Card stamp set (C card 4) Stylized Birthday

Generator Set Controllers) Disabling the generator set. Accidental starting can cause severe injury or death. Before working on the generator set or equipment connected to the set, disable the generator set as follows: (1) Press the generator set off/reset button to shut down the generator set. (2) Disconnectthepowertothebattery charger, if .

41 Hand Reamer adjustable cover max 9,12,18mm-set of 3 01 set 42 Hand Reamer taper 4-9 mm set of 6 or 4-7mm set of 4 02 set 43 Reamer parallel 12-16 mm set of 5 02 no 44 Scraper flat 15 cm 12 nos. 45 Scraper 3 corner 15 cm 12 nos. 46 Spanner adjustable 15 cm 04 set 47 scraper half round 15 cm 12 nos. 48 Chisel cold 9

To Use as a Kitchen Timer ( page 24) Press once Set the kitchen time. Press once To Set Standing Time ( page 24) Set the desired cooking programme. Press once Press once Set the standing time To Set Delay Start ( page 24) Set the delay time. Set the desired cooking programme. Press once Press once F0003BW40QP_OI_08_170630.indd 5 2017/6/30 13:28:24

1981 Honda C70 Passport Parts Catalog Page 11 Carburetor Parts List Diagram No. Description Honda Part No. 1 GASKET SET 16010-086-701 2 NEEDLE SET, JET 16012-174-671 3 FLOAT SET 16013-883-005 4 TOP SET 16014-086-305 5 CHAMBER SET, FLOAT 16015-174-671 6 SCREW SET 16016-174-671 7 VALVE SET, THROTTLE 16022-173-004 8 CARBURETOR ASSY.

Set 7: The NEW Ultimate Karaoke Set - Page 11 to 18 Set 8: The NEW Karaoke Top 2000 Page 18 to 26 Set 9: The OLD Ultimate Karaoke Set - Page 26 to 31 And the add on packs Page 31 to 39 Set 10: The Xtreme Collection Page 39 to 50 KARAOKE HITS Complete 27 disc Set - 180 One Less Bell T

Set Menu 31, CAT Rate to a rate you prefer. I use at least a rate of 19200 and prefer 38400. b. Set Menu 32, CAT TOT to 10 which is the default c. Set Menu 33, CAT RTS to Enable which is the default d. Set Menu 62,PSK e. Set Menu 63 PSK Tone, 1000 Hz f. Set Menu 64, 0Hz g. Set Menu 65, 0Hz

OF CSE, ACE Page 5. DISCRETE MATHEMATICAL STRUCTURES 15CS3 6 Empty Set: A set with no elements is called empty set (or null set, or void set ), and is represented by or {}. Note that nothing preven ts a set from possibly being an element of another se t (whic h is not the same as being a subset!). For i n stance

the generator set by an automatic transfer switch, remote start/stop switch,orenginestartcommandfroma remote computer. Disabling the generator set. Accidental starting can cause severe injury or death. Before working on the generator set or equipment connected to the set, disable the generator set as follows: (1) Press the generator set off/reset

TRI TRAINING PROGRAME 12-WEEK OLYMPIC - BEGINNER WWW.GARMIN.PL 5 Three Tech Run 30: Set 1: 10min Z1 Repeat the following 6x: Set 2: 1min Z3 Set 3: 1min Z1 Set 4: 8min Z1 Work on your technique during the pace variation. Maintain a high RPM, stand tall and pick your knees up whether you are running fast or slow. Easy Swim 1.9: Set 1: 200m Z1 Free and Back Mix Set 2: 5x300m Z1 Pull 30sec .

Fuzzy Logic IJCAI2018 Tutorial 1. Crisp set vs. Fuzzy set A traditional crisp set A fuzzy set 2. . A possible fuzzy set short 10. Example II : Fuzzy set 0 1 5ft 11ins 7 ft height . Fuzzy logic begins by borrowing notions from crisp logic, just as

G021 Microeconomics Lecture notes Ian Preston 1 Consumption set and budget set The consumption set X is the set of all conceivable consumption bundles q, usually identified with Rn The budget set B Xis the set of affordable bundles In standard model individuals can purchase unlimited q

Test Se Electron TM 11-6625-274-12 Tube tester Tube TV-7D/U Test Set, Electron TM 11-6625-316-12 Tube tester Tube TV-2/U (Depot only) Capacitance TM 11-2646A Test set Inductance Resistance Test Set AN/URM-90 Crystal Rectifier Test TM 11-1242 Crystal test set Set TS-268E/U Meter Test Set TM 1