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Molecular Biology Today 2001. 2(3): 45-59.

Molecular Genetics Laboratory: DetailedMolecular Genetics Laboratory: DetailedRequirements for Accreditation by theRequirements for Accreditation by theCollege of American PathologistsCollege of American PathologistsMolecular Genetics Laboratory: Detailed Requirements for Accreditation by theMolecular Genetics Laboratory: Detailed Requirements for Accreditation by theCollege of American PathologistsCollege of American Pathologists

Khaled Khader Abu-AmeroKhaled Khader Abu-Amero1,1,* * and Sayeda Nasreen Abu-Ameroand Sayeda Nasreen Abu-Amero22

1Molecular Genetics and DNA Diagnostics Laboratory, King Faisal Specialist Hospital andResearch Center, (MBC # 03), P.O. Box 3354, Riyadh 11211, Saudi Arabia 2Genomics Research Unit, King Faisal Specialist Hospital and Research Center, Riyadh,Saudi Arabia

AbstractAbstract

The Objectives of this review is to assimilate all known requirements in a singleThe Objectives of this review is to assimilate all known requirements in a singlearticle for individuals or article for individuals or organizations interested in accrediting a molecularorganizations interested in accrediting a moleculargenetics laboratory by College of American Pathologists (CAP). The CAPgenetics laboratory by College of American Pathologists (CAP). The CAPchecklists, which are sent to laboratories applying for accreditation, are serieschecklists, which are sent to laboratories applying for accreditation, are seriesof questions designed to interrogate laboratory of questions designed to interrogate laboratory standards and all relatedstandards and all relatedaspects pertaining to quality. However it is by no means a fully detailedaspects pertaining to quality. However it is by no means a fully detailedprotocol to be followed to achieve protocol to be followed to achieve full accreditation, hence the need for thisfull accreditation, hence the need for thisreview, and individuals or organisations are obliged to seek further review, and individuals or organisations are obliged to seek further supportingsupportingdocumentation and literature.documentation and literature.

The accreditation program is dependent upon successful performance in theThe accreditation program is dependent upon successful performance in themolecular genetics survey molecular genetics survey (proficiency testing) for each analyte tested and(proficiency testing) for each analyte tested andpassing the on- site inspection. The on-site inspections are carried out bypassing the on- site inspection. The on-site inspections are carried out bypracticing laboratorians with expertise in molecular genetics, who uses apracticing laboratorians with expertise in molecular genetics, who uses alaboratory general checklist (covering general aspects laboratory general checklist (covering general aspects related to all clinicalrelated to all clinicallaboratories) and molecular pathology checklist (covering specific requirementslaboratories) and molecular pathology checklist (covering specific requirementsfor molecular for molecular genetics). Once deficiencies cited during inspection aregenetics). Once deficiencies cited during inspection arecorrected, the laboratory will be accredited for a two-year period.corrected, the laboratory will be accredited for a two-year period.Accreditation Accreditation is maintained through continued successful participation in theis maintained through continued successful participation in theproficiency testing and completion of a mandatory proficiency testing and completion of a mandatory self-evaluation, which isself-evaluation, which isdone during the second year of the accreditation cycle. Accreditation is denieddone during the second year of the accreditation cycle. Accreditation is deniedwhen the laboratory fails to meet when the laboratory fails to meet the CAP standards for laboratorythe CAP standards for laboratoryaccreditation.accreditation.

IntroductionIntroduction

In recent years the field of molecular genetics has matured dramatically to theIn recent years the field of molecular genetics has matured dramatically to thepoint that techniques involved are now widely used point that techniques involved are now widely used in routine practices.in routine practices.

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Currently, molecular diagnostics is used in most major medical centers andCurrently, molecular diagnostics is used in most major medical centers andnumerous commercial laboratories numerous commercial laboratories -and can provide valuable information which-and can provide valuable information whichimpact positively on the well being of the individual.impact positively on the well being of the individual.

Since the development of this technology, experts called for quality assuranceSince the development of this technology, experts called for quality assurancemeasures, standards and recommendations measures, standards and recommendations governing genetic testing. Thegoverning genetic testing. TheCollege of American Pathologists was amongst the first accreditationCollege of American Pathologists was amongst the first accreditationorganizations to call for establishment organizations to call for establishment of standards in such complex testing.of standards in such complex testing.These quality assurance measures and standards can now be achieved throughThese quality assurance measures and standards can now be achieved throughthe the Laboratory Accreditation Program (LAP) run by CAP.Laboratory Accreditation Program (LAP) run by CAP.

The history of LAP goes back to 1961, when it was first initiated by CAP and inThe history of LAP goes back to 1961, when it was first initiated by CAP and in1967, the U.S. Clinical Laboratory Improvement 1967, the U.S. Clinical Laboratory Improvement Act (CLIA) came into effect,Act (CLIA) came into effect,which recognized laboratories accredited by CAP. In 1970 the Jointwhich recognized laboratories accredited by CAP. In 1970 the JointCommission on Accreditation of Commission on Accreditation of Healthcare Organizations recognized the LAPHealthcare Organizations recognized the LAPoffered by CAP, resulting in a large number of laboratories entering theoffered by CAP, resulting in a large number of laboratories entering theprogram (Hamlin program (Hamlin and Duckworth, 1997). LAP is voluntary in that eachand Duckworth, 1997). LAP is voluntary in that eachlaboratory desiring accreditation must request it. LAP is widely recognized aslaboratory desiring accreditation must request it. LAP is widely recognized asthe the "gold standard" of laboratory accreditation programs and has served as a"gold standard" of laboratory accreditation programs and has served as amodel for various state and private accreditation programs model for various state and private accreditation programs throughout thethroughout theworld. In fact, several governmental regulatory agencies (e.g. the U.S. Healthworld. In fact, several governmental regulatory agencies (e.g. the U.S. HealthCare Financing Agency) as well as private Care Financing Agency) as well as private agencies (e.g. The Joint Commissionagencies (e.g. The Joint Commissionon Accreditation of Health Care Organizations) accept the LAP in place of theiron Accreditation of Health Care Organizations) accept the LAP in place of theirown programs for own programs for laboratory accreditation. The program has now accreditedlaboratory accreditation. The program has now accreditedmore than 6,000 laboratories. Although the majority of laboratories accreditedmore than 6,000 laboratories. Although the majority of laboratories accreditedare are in the USA or Canada the program has accredited several laboratoriesin the USA or Canada the program has accredited several laboratoriesaround the world (Merrick, 2000).around the world (Merrick, 2000).

The program examines all aspects of quality control and quality improvement inThe program examines all aspects of quality control and quality improvement inthe field of molecular genetics, including the field of molecular genetics, including test methodologies, reagents, controltest methodologies, reagents, controlmedia, equipment, specimen handling, procedure manuals, test reporting andmedia, equipment, specimen handling, procedure manuals, test reporting andinternal and internal and external proficiency testing. In addition, the LAP monitors allexternal proficiency testing. In addition, the LAP monitors allaspects related to personnel, safety, laboratory computer services, aspects related to personnel, safety, laboratory computer services, space,space,communications and overall management practices. The LAP uses ancommunications and overall management practices. The LAP uses aneducational, peer-reviewed inspection process, which educational, peer-reviewed inspection process, which allows any laboratory toallows any laboratory tobe inspected by knowledgeable working professionals who are in tune with thebe inspected by knowledgeable working professionals who are in tune with thechanging needs of the changing needs of the laboratory community. This serves the purpose oflaboratory community. This serves the purpose ofadding an educational experience to the inspection process and allows bothadding an educational experience to the inspection process and allows bothinspectors inspectors and laboratory staff to share their knowledge and expertiseand laboratory staff to share their knowledge and expertise(Merrick, 2000).(Merrick, 2000).

This review offers detailed updated requirements for accrediting molecularThis review offers detailed updated requirements for accrediting moleculargenetics laboratories by the Laboratory genetics laboratories by the Laboratory Accreditation Program of the CollegeAccreditation Program of the Collegeof American Pathologists. The review covers the accreditation requirements ofof American Pathologists. The review covers the accreditation requirements ofmolecular diagnostic molecular diagnostic methods for genetic diseases but not that for infectiousmethods for genetic diseases but not that for infectiousdiseases. Additionally, the overall process of accreditation by CAP will not bediseases. Additionally, the overall process of accreditation by CAP will not bediscussed here as it has been reviewed elsewhere discussed here as it has been reviewed elsewhere (Abu-Amero(Abu-Amero et al et al., 2001).., 2001).

We hope that this review will be useful to professionals working in the field ofWe hope that this review will be useful to professionals working in the field of

molecular genetics and to others molecular genetics and to others considering accreditation by CAP. Althoughconsidering accreditation by CAP. Althoughmost accreditation requirements listed here are those for CAP, readers who aremost accreditation requirements listed here are those for CAP, readers who areseeking seeking accreditation by other agencies may find this review helpful. Readersaccreditation by other agencies may find this review helpful. Readersshould note that the CAP requirements for accreditation are constantly should note that the CAP requirements for accreditation are constantly beingbeingupdated and hence it is necessary to contact the CAP office to ensureupdated and hence it is necessary to contact the CAP office to ensurecompliance with the most up to date requirements.compliance with the most up to date requirements.

Accreditation Requirements for Molecular Genetics LaboratoriesAccreditation Requirements for Molecular Genetics Laboratories

CAP requirements for most operations carried out by molecular geneticCAP requirements for most operations carried out by molecular geneticdiagnostic laboratories will be discussed below.diagnostic laboratories will be discussed below.

1. Requisitions and Specimen Receipt1. Requisitions and Specimen Receipt

All specimens should be accompanied by a requisition form which contains asAll specimens should be accompanied by a requisition form which contains asmuch of the following information as possible: much of the following information as possible: unique patient identification,unique patient identification,sex, date and time of specimen collection, specimen type, race/ethnicity,sex, date and time of specimen collection, specimen type, race/ethnicity,unique identifier found on the unique identifier found on the specimen container, tests requested, patientspecimen container, tests requested, patientlocation, reason for requesting the test, relevant clinical or laboratorylocation, reason for requesting the test, relevant clinical or laboratoryinformation, pedigree information, pedigree (required for linkage analysis, recommended for all(required for linkage analysis, recommended for allcases), referring physician or health professional and billing information.cases), referring physician or health professional and billing information.

All specimens received should be uniquely identified to minimize sample mix-All specimens received should be uniquely identified to minimize sample mix-ups, mislabeling etc. The system should allow ups, mislabeling etc. The system should allow to positively identifying allto positively identifying allpatient specimens, specimen type and aliquots at all times. A bar codingpatient specimens, specimen type and aliquots at all times. A bar codingsystem is recommended, which system is recommended, which will also help to ensure confidentiality.will also help to ensure confidentiality.

2. Specimen Handling2. Specimen Handling

The laboratory must have adequate instructions for specimen collection andThe laboratory must have adequate instructions for specimen collection andhandling before being received by the laboratory. handling before being received by the laboratory. These instructions will be forThese instructions will be forproper labeling of specimens, proper collection of specimens from all relevantproper labeling of specimens, proper collection of specimens from all relevantsources, delivery of sources, delivery of specimens, specimen preservation if processing will bespecimens, specimen preservation if processing will bedelayed (e.g., refrigeration) and procedure for safe handling of specimens. Alldelayed (e.g., refrigeration) and procedure for safe handling of specimens. Allspecimens received should be recorded in an accession book, worksheet,specimens received should be recorded in an accession book, worksheet,computer or other comparable record together with the date and time computer or other comparable record together with the date and time ofofreceipt.receipt.

There should be a written criteria for rejection of unacceptable specimens. ForThere should be a written criteria for rejection of unacceptable specimens. Forevery test offered, documentation every test offered, documentation describing appropriate and inappropriatedescribing appropriate and inappropriateclinical indications and the procedure for rejection of irrelevant samples shouldclinical indications and the procedure for rejection of irrelevant samples shouldbe in place. There be in place. There should be a written policy that no aliquot is ever returned toshould be a written policy that no aliquot is ever returned tothe original container. Similarly a written procedure should be in place for the original container. Similarly a written procedure should be in place for safesafealiquoting of samples in a way to prevent cross-contamination and schedule foraliquoting of samples in a way to prevent cross-contamination and schedule forretaining specimens.retaining specimens.

For chorionic villi or amniotic fluid cells, there should be a back up cell cultureFor chorionic villi or amniotic fluid cells, there should be a back up cell cultureavailable. The molecular genetic laboratory does available. The molecular genetic laboratory does not necessarily need to benot necessarily need to beresponsible for the cell culture work provided that additional material forresponsible for the cell culture work provided that additional material fortesting is readily available if required.testing is readily available if required.

2.1 Parentage and Forensic Identity Testing2.1 Parentage and Forensic Identity Testing

The following regulation should be strictly adhered to when handling theseThe following regulation should be strictly adhered to when handling thesetypes of specimens. Verified identification of all types of specimens. Verified identification of all individuals presentingindividuals presentingthemselves for testing should be documented (the use of photographs and/orthemselves for testing should be documented (the use of photographs and/orfingerprints is strongly fingerprints is strongly recommended). Procedures should be adequate torecommended). Procedures should be adequate toverify specimen identity, integrity and to maintain chainofcustody throughoutverify specimen identity, integrity and to maintain chainofcustody throughoutall steps of all steps of the process beginning with specimen collection including packagingthe process beginning with specimen collection including packagingand transportation. Any tampering with the specimens upon arrival and transportation. Any tampering with the specimens upon arrival at theat thelaboratory should be documented. In addition, specimens should be maintainedlaboratory should be documented. In addition, specimens should be maintainedin a secured area with limited access at all in a secured area with limited access at all times (Tsongalis times (Tsongalis et alet al., 1999).., 1999).

3.3. Specimen ProcessingSpecimen Processing

Molecular diagnosis may be accomplished by any of several methodologies. CAPMolecular diagnosis may be accomplished by any of several methodologies. CAPdoes not favor any particular technique over does not favor any particular technique over another of equivalent sensitivityanother of equivalent sensitivityand specificity, provided the laboratory can demonstrate reliable results andand specificity, provided the laboratory can demonstrate reliable results andquality control with quality control with whichever technique is chosen.whichever technique is chosen.

3.1 Sample Identification3.1 Sample Identification

Sample identification should be assured through all applicable phases ofSample identification should be assured through all applicable phases ofanalysis including nucleic acid extraction and analysis including nucleic acid extraction and quantification, restriction enzymequantification, restriction enzymedigest, electrophoresis, transfer, hybridization, detection, digest, electrophoresis, transfer, hybridization, detection, in situin situ hybridization, hybridization,enzymatic amplification, enzymatic amplification, photography and storage.photography and storage.

3.2 Nucleic Acids Extraction3.2 Nucleic Acids Extraction

Nucleic acids should be extracted and purified by methods reported in theNucleic acids should be extracted and purified by methods reported in theliterature; if not there should be documented evaluation literature; if not there should be documented evaluation of the method used.of the method used.Extracted nucleic acids should be stored in a manner adequate to preventExtracted nucleic acids should be stored in a manner adequate to preventdegradation. Isolated DNA should degradation. Isolated DNA should be stored in a tightly capped container andbe stored in a tightly capped container andkept at 4°C (stability of DNA can be guaranteed for many months at thiskept at 4°C (stability of DNA can be guaranteed for many months at thistemperature). temperature). Long-term storage should be carried out at -20°C or -70°C toLong-term storage should be carried out at -20°C or -70°C toprevent degradation. RNA should be prevent degradation. RNA should be stored stored at at -20°C -20°C or or -70°C once -70°C once extracted,extracted,since RNA degrades quickly (Brown, 1991b).since RNA degrades quickly (Brown, 1991b).

3.3 Nucleic Acids Quantification3.3 Nucleic Acids Quantification

The quantity of nucleic acid should be measured and recorded. This is usuallyThe quantity of nucleic acid should be measured and recorded. This is usuallydone using a spectrophotometer that has been done using a spectrophotometer that has been properly calibrated with the useproperly calibrated with the useof proper controls and measuring the absorbance. This should be performed inof proper controls and measuring the absorbance. This should be performed inclean, dry, quartz cuvettes clean, dry, quartz cuvettes within the linear range of the particularwithin the linear range of the particularspectrophotometer being used. To determine the concentration of purifiedspectrophotometer being used. To determine the concentration of purifiedDNA, an DNA, an absorbency reading at 260 nm (nucleic acid absorbs maximally at thisabsorbency reading at 260 nm (nucleic acid absorbs maximally at thiswavelength) should be performed. An absorbance reading of 1 wavelength) should be performed. An absorbance reading of 1 corresponds tocorresponds toapproximately 50 mg/ml for dsDNA. Since proteins absorbs maximally at 280approximately 50 mg/ml for dsDNA. Since proteins absorbs maximally at 280nm, determination of the nm, determination of the AA260260/A/A280280 ratio provides a qualitative measurement ratio provides a qualitative measurementof the level of DNA in respect to the amount of contaminating protein in theof the level of DNA in respect to the amount of contaminating protein in thesample. Ratios of 1.8 to sample. Ratios of 1.8 to 2.0 indicate high levels of DNA purity. If the ratio is2.0 indicate high levels of DNA purity. If the ratio is

below 1.6, purity may be improved by re-extraction and precipitation (Brown,below 1.6, purity may be improved by re-extraction and precipitation (Brown,1996).1996).

3.4 Quality of Extracted Nucleic Acids3.4 Quality of Extracted Nucleic Acids

The quality (intactness) of high molecular weight DNA and RNA should beThe quality (intactness) of high molecular weight DNA and RNA should beassessed. The laboratory should carefully follow assessed. The laboratory should carefully follow established protocol andestablished protocol andincorporate controls to verify proper performance for each extraction.incorporate controls to verify proper performance for each extraction.

3.5 PCR Methodologies3.5 PCR Methodologies

3.5.1 Amplification3.5.1 Amplification

To assure PCR product specificity, all reaction To assure PCR product specificity, all reaction conditionsconditions (reagents and(reagents andthermocycling parameters) must be established for each thermocycling parameters) must be established for each test system. Reactiontest system. Reactionconditions must provide the desired degree of PCR product specificity. Whenconditions must provide the desired degree of PCR product specificity. Whenamplification of a variable amplification of a variable lengthlength sequence is assayed, the system should besequence is assayed, the system should betested with DNAs from individuals representing large and small amplificationtested with DNAs from individuals representing large and small amplificationproducts products to evaluate the impact of differential amplification (Brown, 1991a).to evaluate the impact of differential amplification (Brown, 1991a).

3.5.2 PCR Product Detection and Analysis3.5.2 PCR Product Detection and Analysis

Detection systems (visual, restriction site, allele specific oligonucleotide,Detection systems (visual, restriction site, allele specific oligonucleotide,hybridization, etc.) employed in diagnostic testing are hybridization, etc.) employed in diagnostic testing are being rapidly adaptedbeing rapidly adaptedfrom established research and diagnostic protocols. Such systems should befrom established research and diagnostic protocols. Such systems should bewell documented and published. well documented and published. The laboratory must demonstrate that a levelThe laboratory must demonstrate that a levelof specificity characteristic of the selected detection system has beenof specificity characteristic of the selected detection system has beenattained internally attained internally and that the level of specificity is adequate for detectingand that the level of specificity is adequate for detectingthe expected products. Adequate care must be taken to guard against failurethe expected products. Adequate care must be taken to guard against failureto detect PCR products.to detect PCR products.

3.5.3 Controls and Standards3.5.3 Controls and Standards

For each PCR run, three types of controls should be included. A positiveFor each PCR run, three types of controls should be included. A positivecontrol, which will provide specific evidence of amplification control, which will provide specific evidence of amplification for each mutationfor each mutationor genotype tested (positive controls must include individuals of knownor genotype tested (positive controls must include individuals of knowngenotype for the locus being tested); a genotype for the locus being tested); a negative (normal) control which meansnegative (normal) control which meansrunning a DNA sample from a patient screened previously and found to berunning a DNA sample from a patient screened previously and found to benegative for the mutation negative for the mutation or the disease being investigated and a blank controlor the disease being investigated and a blank controlwhich contains all components of an amplification reaction except templatewhich contains all components of an amplification reaction except templateDNA. The primary purpose of this final control is to detect contamination withDNA. The primary purpose of this final control is to detect contamination withDNA, especially amplicons from previous amplification DNA, especially amplicons from previous amplification reactions (Erlich, 1999).reactions (Erlich, 1999).In addition, a known molecular weight marker that spans the range of expectedIn addition, a known molecular weight marker that spans the range of expectedproduct size should be used for product size should be used for each electrophoretic run, which will help ineach electrophoretic run, which will help inestimating the size of the PCR product. Controls for various types of assaysestimating the size of the PCR product. Controls for various types of assaysare as follow:are as follow:

Assays based on presence or absence of PCR products must include an internalAssays based on presence or absence of PCR products must include an internalcontrol yielding a positive result to check control yielding a positive result to check for proper amplification and sizing offor proper amplification and sizing ofthe PCR products and to ensure that a negative result is accurate (Rosenstrausthe PCR products and to ensure that a negative result is accurate (Rosenstraus

et alet al., 1998).., 1998).

When specimens are analyzed for sequence variation (Restriction FragmentWhen specimens are analyzed for sequence variation (Restriction FragmentLength Polymorphisms (RFLP) sites, mutation Length Polymorphisms (RFLP) sites, mutation specific sites, etc.) controlsspecific sites, etc.) controlscontaining all alleles to be detected must be included.containing all alleles to be detected must be included.

Assays in which the result is based on fragment size [Variable Number ofAssays in which the result is based on fragment size [Variable Number ofTandem Repeats (VNTRs), microsatellites, etc.] Tandem Repeats (VNTRs), microsatellites, etc.] must include size markersmust include size markers(sequencing ladders, etc.) covering the range of expected results during gel(sequencing ladders, etc.) covering the range of expected results during gelelectrophoresis.electrophoresis.

Assays based on changes in electrophoretic mobility (homo/heteroduplexAssays based on changes in electrophoretic mobility (homo/heteroduplexanalysis, single strand conformation analysis, analysis, single strand conformation analysis, etc.) must include appropriateetc.) must include appropriatecontrols to ensure correct interpretation of results. Any unexpected resultscontrols to ensure correct interpretation of results. Any unexpected resultsrequire repeat of assay. require repeat of assay. Procedures for analysis of possible new mutationsProcedures for analysis of possible new mutationsshould be available.should be available.

3.6 Restriction Enzyme Digestion3.6 Restriction Enzyme Digestion

Efficiency of restriction endonuclease digestion may be confirmed by includingEfficiency of restriction endonuclease digestion may be confirmed by includingan undigested control sample, which contains an undigested control sample, which contains DNA, restriction enzyme bufferDNA, restriction enzyme bufferand distilled water in the absence of restriction enzyme and electrophoresingand distilled water in the absence of restriction enzyme and electrophoresingalongside digested alongside digested samples. The sum of all fragments sizes of the digestedsamples. The sum of all fragments sizes of the digestedproduct should be equivalent to the size of the undigested fragment (Brown,product should be equivalent to the size of the undigested fragment (Brown,1991c).1991c).

3.7 Denaturing Gradient Gel Electrophoresis (DGGE) Assays3.7 Denaturing Gradient Gel Electrophoresis (DGGE) Assays

3.7.1 PCR Fragment Design3.7.1 PCR Fragment Design

All sequences to be analyzed by DGGE should be amplified by PCR usingAll sequences to be analyzed by DGGE should be amplified by PCR usingprotocols optimized for the amplicon in question. protocols optimized for the amplicon in question. The specificity of the PCRThe specificity of the PCRreaction should be such that a single amplicon is seen on a stained gel. Eachreaction should be such that a single amplicon is seen on a stained gel. Eachamplicon should be designed amplicon should be designed using available software or empiric analysis tousing available software or empiric analysis toproduce a single melting domain throughout the region to be assessed. Theproduce a single melting domain throughout the region to be assessed. Theprimers used primers used in the amplification step should be designed to include a 5'-in the amplification step should be designed to include a 5'-clamp sufficient to stabilize the melting domain of the test DNA clamp sufficient to stabilize the melting domain of the test DNA sequencesequence(Fischer and Lerman, 1983).(Fischer and Lerman, 1983).

3.7.2 Sample Preparation3.7.2 Sample Preparation

DNA samples should be prepared, stored and amplified according to theDNA samples should be prepared, stored and amplified according to thepreviously mentioned guidelines. Samples should be previously mentioned guidelines. Samples should be heated and allowed to re-heated and allowed to re-anneal prior to loading to permit heteroduplex formation. Time andanneal prior to loading to permit heteroduplex formation. Time andtemperature should be standardized. If a temperature should be standardized. If a potential homozygous mutantpotential homozygous mutantcondition is being analyzed, it may be appropriate to mix a known normalcondition is being analyzed, it may be appropriate to mix a known normalcontrol and test sample to control and test sample to force heteroduplex formation.force heteroduplex formation.

3.7.3 Gel Electrophoresis3.7.3 Gel Electrophoresis

Appropriate denaturing gradient conditions should be established based onAppropriate denaturing gradient conditions should be established based on

calculated melting profile and empiric results observed calculated melting profile and empiric results observed with positive controls.with positive controls.A set of positive controls should include (whenever possible) samplesA set of positive controls should include (whenever possible) samplescontaining mutations distributed throughout containing mutations distributed throughout the region to be analyzed.the region to be analyzed.Equipment used to form the gradients in the gels and to run gels underEquipment used to form the gradients in the gels and to run gels undertemperature-controlled conditions temperature-controlled conditions should be standardized within eachshould be standardized within eachlaboratory. Any change in equipment will require a re-validation of the assay.laboratory. Any change in equipment will require a re-validation of the assay.Samples to be run on Samples to be run on the same gel should be denatured, annealed, and loadedthe same gel should be denatured, annealed, and loadedon the gel at the same on the gel at the same time.time. A positive control sample should be A positive control sample should be analyzedanalyzedsimultaneously to provide a measure of the adequacy of the heteroduplexsimultaneously to provide a measure of the adequacy of the heteroduplexformation and the gel running conditions. A negative formation and the gel running conditions. A negative (normal) control sample(normal) control samplecan be used to aid in sizing of the observed can be used to aid in sizing of the observed bandsbands. . It is not necessary to run aIt is not necessary to run asample of every known mutation in sample of every known mutation in each gel. A single mutation control iseach gel. A single mutation control issufficient to document the reproducibility of the system.sufficient to document the reproducibility of the system.

3.7.4 Data Analysis3.7.4 Data Analysis

Gels should be stained (or visualized based on labeled DNA) in a mannerGels should be stained (or visualized based on labeled DNA) in a manneradequate to detect the entire banding pattern adequate to detect the entire banding pattern created. Heteroduplexes arecreated. Heteroduplexes areoften present in smaller amounts than the homoduplex forms and may produceoften present in smaller amounts than the homoduplex forms and may producea lighter signal. Samples on a lighter signal. Samples on the gels should be identified by an unambiguousthe gels should be identified by an unambiguousmethod clearly identifying positive and negative method clearly identifying positive and negative controls.controls. Documentation of gelDocumentation of gelresults by photography or other image storage system is necessary.results by photography or other image storage system is necessary.Computerized image analysis may be helpful in identification of Computerized image analysis may be helpful in identification of recurringrecurringmutations. The presence of putative mutations identified by DGGE must bemutations. The presence of putative mutations identified by DGGE must beconfirmed by sequencing.confirmed by sequencing.

3.7.5 Validation3.7.5 Validation

Each laboratory must validate the technique for each sequence to be analyzed.Each laboratory must validate the technique for each sequence to be analyzed.Validation with known mutations as well as Validation with known mutations as well as normal samples is required. Resultsnormal samples is required. Resultsof validation studies for each gene analyzed must be available for review.of validation studies for each gene analyzed must be available for review.

3.8 Heteroduplex Assays3.8 Heteroduplex Assays

PCR product sizes of approximately 150-300 bp are ideal for screeningPCR product sizes of approximately 150-300 bp are ideal for screeningunknown mutations by heteroduplex analysis. Larger unknown mutations by heteroduplex analysis. Larger fragments can be used tofragments can be used todetect specific mutations or polymorphisms once it has been established that adetect specific mutations or polymorphisms once it has been established that aheteroduplex band can be heteroduplex band can be consistently detected under standardized conditions.consistently detected under standardized conditions.The location of the mutation/polymorphism of interest should be at least 40-The location of the mutation/polymorphism of interest should be at least 40-50 bases from 50 bases from the ends of the DNA fragments. Thus, PCR primers in flankingthe ends of the DNA fragments. Thus, PCR primers in flankingintron sequences should be at 40-50 bases from the intron-exon intron sequences should be at 40-50 bases from the intron-exon junctions. PCRjunctions. PCRamplification of the regions of interest should be carried out according to allamplification of the regions of interest should be carried out according to allstandard precautions. It is critical that each standard precautions. It is critical that each amplicon produce a clean, singleamplicon produce a clean, singleband for use in heteroduplex analysis. Samples should be heat denatured andband for use in heteroduplex analysis. Samples should be heat denatured andallowed to re-anneal to allowed to re-anneal to facilitate heteroduplex formation. The time andfacilitate heteroduplex formation. The time andtemperature for denaturation and annealing should be standardized. In case oftemperature for denaturation and annealing should be standardized. In case ofpotential homozygous mutations, PCR products from wild type controls shouldpotential homozygous mutations, PCR products from wild type controls shouldbe mixed, denatured and re-annealed with the test samples be mixed, denatured and re-annealed with the test samples to force theto force theformation of heteroduplexes. The composition of the gel matrix to be used forformation of heteroduplexes. The composition of the gel matrix to be used forheteroduplex analysis, the thickness of the heteroduplex analysis, the thickness of the gel, the length and time of the run,gel, the length and time of the run,

and the electrophoresis equipment should be standardized within eachand the electrophoresis equipment should be standardized within eachlaboratory. Samples to laboratory. Samples to be analyzed on the same gel should be denatured, re-be analyzed on the same gel should be denatured, re-annealed and loaded on the gel run to validate the results for each gel.annealed and loaded on the gel run to validate the results for each gel.Heteroduplex gels should be visualized by staining or by autoradiography,Heteroduplex gels should be visualized by staining or by autoradiography,depending on the detection system employed, to detect the entire depending on the detection system employed, to detect the entire bandingbandingpattern required for mutation detection. The detection system used to detectpattern required for mutation detection. The detection system used to detectthe heteroduplex bands (e.g., the specific staining the heteroduplex bands (e.g., the specific staining protocol) should beprotocol) should bestandardized in each laboratory. Results should be scored unambiguously bystandardized in each laboratory. Results should be scored unambiguously bycomparison with the positive and comparison with the positive and negative controls. All putative positivenegative controls. All putative positiveresults detected by heteroduplex analysis should be confirmed by sequencingresults detected by heteroduplex analysis should be confirmed by sequencingto identify the mutation to identify the mutation or polymorphism involved. The heteroduplex analysisor polymorphism involved. The heteroduplex analysistechnique should be validated by using known mutations, which should technique should be validated by using known mutations, which should exhibitexhibitdetectable and in many cases characteristic heteroduplex banding patterns fordetectable and in many cases characteristic heteroduplex banding patterns forspecific mutations, as well as normal control specific mutations, as well as normal control samples. For each gene analyzedsamples. For each gene analyzedby heteroduplex analysis, validation test results should be available for reviewby heteroduplex analysis, validation test results should be available for review(Glavic and Dean, 1995).(Glavic and Dean, 1995).

3.9 Southern Analysis3.9 Southern Analysis

3.9.1 Restriction Digestion and Electrophoresis3.9.1 Restriction Digestion and Electrophoresis

Restriction endonuclease digestion of prepared DNA for Southern analysis mustRestriction endonuclease digestion of prepared DNA for Southern analysis mustbe done according to a standardized protocol, be done according to a standardized protocol, which will be documented in thewhich will be documented in thelaboratory manual.laboratory manual.

Quality control of restriction digests must be done by one of the following: RunQuality control of restriction digests must be done by one of the following: Runa test gel prior to electrophoresis. If incomplete, a test gel prior to electrophoresis. If incomplete, re-digest the specimen.re-digest the specimen.Evaluate the analytical gel by visually comparing to size markers or to theEvaluate the analytical gel by visually comparing to size markers or to thepatterns of all DNAs on the patterns of all DNAs on the gel, including controls, for consistency of satellitegel, including controls, for consistency of satellitebands as well as high and low molecular weight bands. Each test must includebands as well as high and low molecular weight bands. Each test must includehuman DNA control(s) with documented genotype at the locus tested (Brown,human DNA control(s) with documented genotype at the locus tested (Brown,1993).1993).

3.9.2 Membrane Preparation3.9.2 Membrane Preparation

Prior to transfer, the Southern gel must be photographed to provide a hardPrior to transfer, the Southern gel must be photographed to provide a hardcopy documentation of the gel. The method of transfer copy documentation of the gel. The method of transfer must be documented inmust be documented inthe laboratory manual with appropriate references. Efficiency of transfer mustthe laboratory manual with appropriate references. Efficiency of transfer mustbe validated and documented be validated and documented either at time of transfer or at the end of theeither at time of transfer or at the end of thestudy by using photographic or autoradiographic film and appropriate controlstudy by using photographic or autoradiographic film and appropriate controlDNA, DNA, including human control(s), digested along side the samples. All Southernincluding human control(s), digested along side the samples. All Southerngels should include internal and external size markers to assist in gels should include internal and external size markers to assist in the readingthe readingof the alleles. External markers may be excluded if appropriate heterozygotesof the alleles. External markers may be excluded if appropriate heterozygotesor "all allele" controls are used.or "all allele" controls are used.

3.9.3 Hybridization3.9.3 Hybridization

Hybridizations must be carried out by accepted procedures and documentedHybridizations must be carried out by accepted procedures and documentedwith appropriate references. Hybridization can be with appropriate references. Hybridization can be checked by scoring thechecked by scoring theknown controls included on the Southern filter. For those markers new to theknown controls included on the Southern filter. For those markers new to the

laboratory, a previously used filter, laboratory, a previously used filter, if available, on which the DNA has been cutif available, on which the DNA has been cutwith the appropriate enzyme (or a test DNA of known genotype), shall be usedwith the appropriate enzyme (or a test DNA of known genotype), shall be usedas as further quality control of the hybridization. The laboratory must retain afurther quality control of the hybridization. The laboratory must retain arepresentation of the primary data (gel, film, autoradiograph, representation of the primary data (gel, film, autoradiograph, etc.)etc.)demonstrating the reported hybridization pattern.demonstrating the reported hybridization pattern.

3.10 Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis (SDS-PAGE)3.10 Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Translation products are separated by discontinuous SDS-PAGE. CommerciallyTranslation products are separated by discontinuous SDS-PAGE. Commerciallyavailable protein markers are usually used as available protein markers are usually used as molecular size standards. If themolecular size standards. If theprotein product of interest is very large, special standards may be required. Aprotein product of interest is very large, special standards may be required. Anormal control must be run normal control must be run with each batch of test samples. Previouslywith each batch of test samples. Previouslyprepared (known product size) controls may be used as an external sizeprepared (known product size) controls may be used as an external sizeindicator, but indicator, but a simultaneously transcribed/translated control is also requireda simultaneously transcribed/translated control is also required(Maniatis (Maniatis et alet al., 1989a).., 1989a).

3.10.1 Interpretation3.10.1 Interpretation

A mutation is indicated by the presence of a novel band of lower-than-normalA mutation is indicated by the presence of a novel band of lower-than-normalmolecular weight representing a truncated peptide. If molecular weight representing a truncated peptide. If the band representingthe band representingthe full-length polypeptide is present in the same sample, it can serve as anthe full-length polypeptide is present in the same sample, it can serve as aninternal control. Background" bands internal control. Background" bands are often observed. Some of these areare often observed. Some of these areartifacts due to translation from internal AUG codons downstream from theartifacts due to translation from internal AUG codons downstream from theauthentic start codon authentic start codon or erroneous translation termination due to a non-or erroneous translation termination due to a non-optimized optimized ""in vitroin vitro" system. Other background bands present may represent" system. Other background bands present may representproteins proteins in the reticulocyte lysate or alternatively-spliced products from thein the reticulocyte lysate or alternatively-spliced products from thegene of interest. Again, comparison of bands with those from a gene of interest. Again, comparison of bands with those from a known normalknown normalcontrol assayed simultaneously is essential. The presence of a truncatedcontrol assayed simultaneously is essential. The presence of a truncatedpolypeptide is suggestive of an underlying polypeptide is suggestive of an underlying genomic mutation. In most cases,genomic mutation. In most cases,the length of the truncated polypeptide (determined by using the proteinthe length of the truncated polypeptide (determined by using the proteinmarkers as standards) can be markers as standards) can be used to localize the putative mutation. If theused to localize the putative mutation. If thepolypeptide is truncated due to a large deletion, the deletion site can bepolypeptide is truncated due to a large deletion, the deletion site can bedetermined by determined by restriction endonuclease mapping. The analytical specificity andrestriction endonuclease mapping. The analytical specificity andsensitivity of the protein truncation assay is not known. It is essential to verifysensitivity of the protein truncation assay is not known. It is essential to verifythe presence of each mutation by either sequencing genomic DNA orthe presence of each mutation by either sequencing genomic DNA orsequencing cDNA followed by analysis of genomic DNA using sequencing cDNA followed by analysis of genomic DNA using RFLP or AlleleRFLP or AlleleSpecific Oligonucleotide (ASO) methodologies.Specific Oligonucleotide (ASO) methodologies.


Each laboratory must validate the technique for each gene to be analyzed.Each laboratory must validate the technique for each gene to be analyzed.Validation with known mutations as well as normal Validation with known mutations as well as normal samples is required. Resultssamples is required. Resultsof validation studies for each gene analyzed must be available for review.of validation studies for each gene analyzed must be available for review.

3.11 DNA Sequencing Analysis3.11 DNA Sequencing Analysis

Although the sequence assay shares elements in common with all other DNAAlthough the sequence assay shares elements in common with all other DNAdiagnostic assays, there are unique concerns and diagnostic assays, there are unique concerns and areas that require separateareas that require separateattention. Unique issues that arise in DNA sequence assays result from theattention. Unique issues that arise in DNA sequence assays result from thelarge number of analytical large number of analytical points measured in each particular assay (i.e., thepoints measured in each particular assay (i.e., the

number of bases analyzed) and the relatively small signal strengths that arenumber of bases analyzed) and the relatively small signal strengths that areobtained obtained from any base at any position. The technology for the generation offrom any base at any position. The technology for the generation ofthe sequence information is also generally complicated. Therefore, the sequence information is also generally complicated. Therefore, thethesequence information must be verified and controlled at multiple points in thesequence information must be verified and controlled at multiple points in thegeneration and interpretation of the sequencing generation and interpretation of the sequencing data. One very positive aspectdata. One very positive aspectof the emerging use of sequencing for molecular diagnostics is that the likelyof the emerging use of sequencing for molecular diagnostics is that the likelyerrors will be biased errors will be biased very strongly towards the generation of false positives,very strongly towards the generation of false positives,rather than false negatives. This is a consequence of the fact that it is muchrather than false negatives. This is a consequence of the fact that it is mucheasier to produce a sequence that looks as if it contains the wrong base(s)easier to produce a sequence that looks as if it contains the wrong base(s)than a clear profile showing only the correct base. As each than a clear profile showing only the correct base. As each positive can andpositive can andshould be tested by an independent determination, this direction of bias isshould be tested by an independent determination, this direction of bias isdesirable. Potential for missing a heterozygous desirable. Potential for missing a heterozygous base substitution is a concern.base substitution is a concern.To increase the sensitivity of heterozygote detection, both the sequencingTo increase the sensitivity of heterozygote detection, both the sequencingchemistry and polymerase chemistry and polymerase used should be optimized to produce uniform peakused should be optimized to produce uniform peakintensities in the case of fluorescent sequencing, since variations can result inintensities in the case of fluorescent sequencing, since variations can result infalse negatives. Both of these scenarios underscore the need to sequence bothfalse negatives. Both of these scenarios underscore the need to sequence bothstrands of the DNA region analyzed to optimize strands of the DNA region analyzed to optimize sensitivity and specificity ofsensitivity and specificity ofthe assay (Maniatis the assay (Maniatis et alet al., 1989b).., 1989b).

3.11.1 Methodologies3.11.1 Methodologies

Presently the most widely used method is the Sanger dideoxy chainPresently the most widely used method is the Sanger dideoxy chaintermination, which can be applied in several forms. termination, which can be applied in several forms. Manual sequencing requiresManual sequencing requiresa radioactive label a radioactive label ((3232P, P, 3333P or P or 3535S) in one of the four dNTPs or at the 5´ endS) in one of the four dNTPs or at the 5´ endof a sequencing primer. of a sequencing primer. The advantages over automated sequencing includeThe advantages over automated sequencing includegood signal-to-noise ratio. However, the disadvantages are low throughput good signal-to-noise ratio. However, the disadvantages are low throughput andandrequirement for radioactivity. Both manual and computer-assisted readingrequirement for radioactivity. Both manual and computer-assisted readingformats can be used, but computerized systems formats can be used, but computerized systems provide more accurateprovide more accuratetransfer of data. Fluorescent sequencing reactions can be performed using dyetransfer of data. Fluorescent sequencing reactions can be performed using dyeprimers or dye-labeled primers or primers or dye-labeled primers or dye terminator chemistries and one ofdye terminator chemistries and one ofseveral polymerases. Data collection uses an imaging system and appropriateseveral polymerases. Data collection uses an imaging system and appropriatesoftware. software. Automated fluorescent sequencing can be performed usingAutomated fluorescent sequencing can be performed usingautomated sequencer formats providing automated gel running and dataautomated sequencer formats providing automated gel running and datacollection. Capillary gel electrophoresis for sequencing has been described andcollection. Capillary gel electrophoresis for sequencing has been described andis superseding all currently used techniques.is superseding all currently used techniques.

3.11.2 PCR Amplification3.11.2 PCR Amplification

The length of the region to be sequenced in a single run must be limited. AnThe length of the region to be sequenced in a single run must be limited. Anupper limit of accurately readable sequence exists for upper limit of accurately readable sequence exists for each methodology andeach methodology andgel apparatus type. The quantity of the DNA must be sufficient to generategel apparatus type. The quantity of the DNA must be sufficient to generateadequate PCR product. This can adequate PCR product. This can be determined by meeting an expectation ofbe determined by meeting an expectation ofPCR efficiency (e.g., an agarose or acrylamide gel separation of an aliquot ofPCR efficiency (e.g., an agarose or acrylamide gel separation of an aliquot ofthe PCR can the PCR can be compared to a standard).be compared to a standard).

3.11.3 Sanger Sequencing3.11.3 Sanger Sequencing

Primers directed towards the end of the fragments are used. There are severalPrimers directed towards the end of the fragments are used. There are severalchemistries available but each should be aimed chemistries available but each should be aimed at providing the best possibleat providing the best possible

sequence coverage of the fragment.sequence coverage of the fragment.

3.11.4 Gel Electrophoresis3.11.4 Gel Electrophoresis

Following the Sanger reaction, materials must be pooled (dye primer reactions)Following the Sanger reaction, materials must be pooled (dye primer reactions)or purified from unincorporated materials. Normal or purified from unincorporated materials. Normal care is needed to preventcare is needed to preventsample mix-up. The tracking of individual samples on gels is a difficult andsample mix-up. The tracking of individual samples on gels is a difficult andpotentially error-prone step. potentially error-prone step. Standard loading formats should be used toStandard loading formats should be used toensure this part of the process is accurate. Gel preparation using commerciallyensure this part of the process is accurate. Gel preparation using commerciallyavailable available premixed solutions may provide additional quality control. If thepremixed solutions may provide additional quality control. If thesupplier of the solutions changes, separation characteristics must be supplier of the solutions changes, separation characteristics must be re-re-evaluated. The characteristics of each gel apparatus/power supply combinationevaluated. The characteristics of each gel apparatus/power supply combinationare unique. Therefore timing, voltage requirements and are unique. Therefore timing, voltage requirements and separationseparationcharacteristics must be standardized for each individual set-up.characteristics must be standardized for each individual set-up.

3.11.5 Primary Base Calling3.11.5 Primary Base Calling

The overall quality of the sequence reactions must be monitored. The concernThe overall quality of the sequence reactions must be monitored. The concernis that poor sequence reactions containing artifacts is that poor sequence reactions containing artifacts such as "stops,"such as "stops,"compressions, or "Ns" will be difficult to interpret and will result in thecompressions, or "Ns" will be difficult to interpret and will result in theclassification of normal bases as mutant or vice classification of normal bases as mutant or vice versa. Every effort should beversa. Every effort should bemade to resolve any such regions. Routine analysis of the opposite strandmade to resolve any such regions. Routine analysis of the opposite strandsequence will be useful for sequence will be useful for that purpose. The use of a different sequencingthat purpose. The use of a different sequencingchemistry or polymerase may resolve specific regions, since artifacts may notchemistry or polymerase may resolve specific regions, since artifacts may notoccur occur in identical spots under alternate conditions. Currently available criteriain identical spots under alternate conditions. Currently available criteriainclude the number of positions at which computer base calling include the number of positions at which computer base calling is not possible.is not possible.

A comparison of each test with a known standard (e.g., Gene bank) is required,A comparison of each test with a known standard (e.g., Gene bank) is required,including judgment of peak height. including judgment of peak height. (Caution should be exercised, since not all(Caution should be exercised, since not allsequences in Gene bank are correct.). Manual re-reading of areas where thesequences in Gene bank are correct.). Manual re-reading of areas where thesoftware has software has had difficulty should be performed with caution. Thehad difficulty should be performed with caution. Thechromatograms of both the forward and reverse strands should be evaluatedchromatograms of both the forward and reverse strands should be evaluatedand and the consensus compared to the standard sequence.the consensus compared to the standard sequence.

3.11.6 Comparison of Sequence Data with a " Within Run" Standard3.11.6 Comparison of Sequence Data with a " Within Run" Standard

The comparison with a standard of a high quality sequence from the same runThe comparison with a standard of a high quality sequence from the same runis also needed to identify base differences. is also needed to identify base differences. Verification of readings usingVerification of readings usingsecond strand and/or second aliquot sequencing is required. Some mutationssecond strand and/or second aliquot sequencing is required. Some mutationsmay be missed if sequencing may be missed if sequencing is performed in only one direction. Any positivesis performed in only one direction. Any positivesshould be confirmed by sequencing a second aliquot. For direct sequencing, ashould be confirmed by sequencing a second aliquot. For direct sequencing, asecond PCR amplification product should be used for repeat sequence analysis.second PCR amplification product should be used for repeat sequence analysis.

3.11.7 Interpretation and Data Reporting3.11.7 Interpretation and Data Reporting

Base differences are correlated with the known gene structure and otherBase differences are correlated with the known gene structure and otherrelevant data and the likely effect of the base change on relevant data and the likely effect of the base change on the gene is predicted.the gene is predicted.The report should note the exact base change and location by nucleotideThe report should note the exact base change and location by nucleotideposition as referenced in Gene bank position as referenced in Gene bank and the corresponding position change inand the corresponding position change inthe protein using standard nomenclature. For small deletions and insertion orthe protein using standard nomenclature. For small deletions and insertion or

nonsense nonsense mutations resulting in a predicted protein truncation, the termmutations resulting in a predicted protein truncation, the term"mutation" is appropriate. For missense alterations, one must consider "mutation" is appropriate. For missense alterations, one must consider whetherwhetherthese represent mutations, polymorphisms, or rare variants. For each geneticthese represent mutations, polymorphisms, or rare variants. For each geneticdisease, the laboratory should first refer to a disease, the laboratory should first refer to a polymorphism and mutationpolymorphism and mutationdatabase. If the base alteration has not been previously described, the naturedatabase. If the base alteration has not been previously described, the natureand significance of the change may and significance of the change may be unclear and should be stated as such inbe unclear and should be stated as such inthe report. For resolution, family studies and population based studies arethe report. For resolution, family studies and population based studies areappropriate. Reports in which no mutations are detected by sequence analysisappropriate. Reports in which no mutations are detected by sequence analysisshould include multiple disclaimers, primarily that the sensitivity should include multiple disclaimers, primarily that the sensitivity of the test isof the test is<100%. If sequencing was confined to the coding region of the gene, the<100%. If sequencing was confined to the coding region of the gene, thepossibility of mutations in the promoter or possibility of mutations in the promoter or intragenic regions not covered byintragenic regions not covered bythe test should be clearly stated. Sequencing will not detect large genethe test should be clearly stated. Sequencing will not detect large genedeletions or duplications. In addition, deletions or duplications. In addition, a mutation in a different gene thata mutation in a different gene thatcontributes to the disease, as well as misdiagnosis of the proband, constitutecontributes to the disease, as well as misdiagnosis of the proband, constituteother possibilities.other possibilities.



3.12 Single-Strand Conformation Polymorphism (SSCP) Assays3.12 Single-Strand Conformation Polymorphism (SSCP) Assays

3.12.1 Assay Design3.12.1 Assay Design

When screening for unknown mutations, DNA fragments between 150 and 300When screening for unknown mutations, DNA fragments between 150 and 300bp are typically used. Larger fragments can be used bp are typically used. Larger fragments can be used if it is known that theif it is known that thespecific mutation/polymorphism of interest produces an abnormal SSCP patternspecific mutation/polymorphism of interest produces an abnormal SSCP patternin that DNA segment.in that DNA segment.

3.12.2 Polyacrylamide Gel Electrophoresis3.12.2 Polyacrylamide Gel Electrophoresis

Gels should be run for a sufficient length of time (depending on fragmentGels should be run for a sufficient length of time (depending on fragmentlength) to detect possible mobility shifts. In order to reduce length) to detect possible mobility shifts. In order to reduce the risk of missingthe risk of missingmutations, samples should be run under two electrophoretic conditions thatmutations, samples should be run under two electrophoretic conditions thatmay differ in length of time, may differ in length of time, temperature, buffer concentration, cross-linkingtemperature, buffer concentration, cross-linkingratio, cross-linking reagents, and presence or absence of glycerol. It isratio, cross-linking reagents, and presence or absence of glycerol. It ispreferable to preferable to standardize electrophoretic conditions for as many differentstandardize electrophoretic conditions for as many differentmutations as possible. This can be done by using more than one controlmutations as possible. This can be done by using more than one controlmutation (Orita, mutation (Orita, et alet al., 1989).., 1989).

3.12.3 Controls3.12.3 Controls

Double-stranded DNA control should be run alongside single-strandedDouble-stranded DNA control should be run alongside single-strandedfragments to allow identification of both fragments. fragments to allow identification of both fragments. Some mobility shifts areSome mobility shifts areobserved only with double-stranded fragments. Optimal denaturation of double-observed only with double-stranded fragments. Optimal denaturation of double-stranded fragments should involve stranded fragments should involve a dilution of the PCR product. This willa dilution of the PCR product. This will

necessitate use of a sensitive detection method (fluorescence, radioactivity, ornecessitate use of a sensitive detection method (fluorescence, radioactivity, orsilver staining). silver staining). The PCR product from at least one normal control should beThe PCR product from at least one normal control should beincluded on every SSCP gel. The PCR product from at least one included on every SSCP gel. The PCR product from at least one control samplecontrol samplecontaining a mutation should be included on each SSCP gel in order to ensurecontaining a mutation should be included on each SSCP gel in order to ensurethat the electrophoresis conditions are that the electrophoresis conditions are optimal for detection of at least oneoptimal for detection of at least onemutation. Inclusion of more than one control mutation is advisable to improvemutation. Inclusion of more than one control mutation is advisable to improvethe accuracy and the accuracy and standardization of the assay. If screening for several knownstandardization of the assay. If screening for several knownmutations in a DNA fragment, use of control samples for each is desirable tomutations in a DNA fragment, use of control samples for each is desirable toensure that ensure that the sequence alteration produces an abnormal SSCP band underthe sequence alteration produces an abnormal SSCP band underthe conditions used (Orita, the conditions used (Orita, et alet al., 1989).., 1989).

3.12.4 Visualization of Results3.12.4 Visualization of Results

For manual approaches to SSCP using For manual approaches to SSCP using 3232P-labeled or P-labeled or 3333P-labeledP-labeleddeoxynucleotides, multiple X-ray film exposures are deoxynucleotides, multiple X-ray film exposures are recommended to visualizerecommended to visualizeall signals. Some abnormal SSCP bands may be faint, requiring longer exposuresall signals. Some abnormal SSCP bands may be faint, requiring longer exposuresthan normal bands. For SSCP than normal bands. For SSCP by automated fluorescent analysis, internal sizeby automated fluorescent analysis, internal sizemarkers help prevent artifactual lane shifting from influencing mobility shiftmarkers help prevent artifactual lane shifting from influencing mobility shiftdata. It may data. It may be necessary to adjust the volume of sample loaded to achievebe necessary to adjust the volume of sample loaded to achievedetection.detection.

3.12.5 Interpretation of Results3.12.5 Interpretation of Results

All samples showing a mobility shift should be sequenced to determine theAll samples showing a mobility shift should be sequenced to determine thenature of the sequence change. It is possible for nature of the sequence change. It is possible for different sequence variationsdifferent sequence variationsto produce similar SSCP results.to produce similar SSCP results.



3.13 Probe/Primer/Locus Documentation3.13 Probe/Primer/Locus Documentation

All loci used for analysis in the laboratory need to be well documented byAll loci used for analysis in the laboratory need to be well documented byHuman Gene Mapping Workshop, Geneatlas, Genome Human Gene Mapping Workshop, Geneatlas, Genome Data Base (GDB) or byData Base (GDB) or bypublication in the peer-reviewed scientific literature. This documentation mustpublication in the peer-reviewed scientific literature. This documentation mustbe maintained in an be maintained in an up-to-date laboratory book and include the following:up-to-date laboratory book and include the following:genome location, linkage data, literature references, cloning vector, cloninggenome location, linkage data, literature references, cloning vector, cloningsite, size site, size of insert, enzyme used for the detection of the RFLP, the sizes of theof insert, enzyme used for the detection of the RFLP, the sizes of thealleles and any constant bands, the allele frequencies in each alleles and any constant bands, the allele frequencies in each racial or ethnicracial or ethnicgroup for which this information exists, new mutation rate (if known), how thegroup for which this information exists, new mutation rate (if known), how theprobe was prepared as well as hybridization probe was prepared as well as hybridization and wash conditions. Forand wash conditions. Foroligonucleotide probes or primers, documentation sheets also must includeoligonucleotide probes or primers, documentation sheets also must includespecific sequences. For specific sequences. For primers, PCR conditions and the size of the expectedprimers, PCR conditions and the size of the expectedpositive result should be included. There must be internal documentation thatpositive result should be included. There must be internal documentation thatthe the probe/primer used is consistent with the above data (i.e., a photographprobe/primer used is consistent with the above data (i.e., a photographindicating that the size of the insert isolated from the vector is indicating that the size of the insert isolated from the vector is the correctthe correctsize or that the conditions used by the laboratory produce the appropriatesize or that the conditions used by the laboratory produce the appropriate

result) (ACMG, 1999).result) (ACMG, 1999).

3.14 Linkage Analysis3.14 Linkage Analysis

The laboratory must keep an up-to-date reference list documenting linkageThe laboratory must keep an up-to-date reference list documenting linkagerelationships (i.e., location relative to locus in relationships (i.e., location relative to locus in question, recombinationquestion, recombinationfractions and/or q values at 95% confidence intervals) for each disorderfractions and/or q values at 95% confidence intervals) for each disorderanalyzed by indirect linkage methods. analyzed by indirect linkage methods. The laboratory must have documentedThe laboratory must have documentedlinkage relationships for all in-house generated probes prior to use in a clinicallinkage relationships for all in-house generated probes prior to use in a clinicalsetting. In order setting. In order for linkage analysis involving probes with for linkage analysis involving probes with significantsignificantrecombination distances from the locus in question to be reported, the analysisrecombination distances from the locus in question to be reported, the analysismust contain data from two informative flanking markers. If this is notmust contain data from two informative flanking markers. If this is notpossible, the reason must be stated so as to indicate that every possible, the reason must be stated so as to indicate that every effort waseffort wasmade to provide such. For linkage analyses made to provide such. For linkage analyses involvinginvolving probes with negligibleprobes with negligiblerecombination distances from the locus in question, recombination distances from the locus in question, it is only necessary to useit is only necessary to useonly one highly informative marker. For each disease specific system examined,only one highly informative marker. For each disease specific system examined,the number of the number of informative markers to be used is dependent upon theinformative markers to be used is dependent upon theinformativeness of each marker, the disease specific recombination frequencyinformativeness of each marker, the disease specific recombination frequencyand and the availability of markers (Ott, 1991).the availability of markers (Ott, 1991).

3.15 Assays Validation3.15 Assays Validation

Each laboratory must validate the analytical performance characteristicsEach laboratory must validate the analytical performance characteristics(sensitivity, specificity, reproducibility) of the technique (sensitivity, specificity, reproducibility) of the technique chosen for analysis ofchosen for analysis ofeach gene. Validation with well-characterized samples is critical. Whereeach gene. Validation with well-characterized samples is critical. Whereavailable, performance characteristics available, performance characteristics should be compared with an existingshould be compared with an existing"gold standard" assay. In the absence of "gold standards" for comparison of"gold standard" assay. In the absence of "gold standards" for comparison ofresults for new assays, results for new assays, the splitting of samples with another laboratory with anthe splitting of samples with another laboratory with anestablished clinical assay may be considered. Documentation of validationestablished clinical assay may be considered. Documentation of validationresults must be available for review.results must be available for review.

4. Reporting of Results4. Reporting of Results

Laboratory reports should be designed to provide patient laboratory dataLaboratory reports should be designed to provide patient laboratory dataeffectively and completely. In general the report effectively and completely. In general the report should include the followingshould include the followinginformation: date of report, name of individual, date of birth, specimeninformation: date of report, name of individual, date of birth, specimencollection date, specimen collection date, specimen accession number or case number, indication foraccession number or case number, indication fortesting, test performed (including mutation tested), brief description of testtesting, test performed (including mutation tested), brief description of testmethodology, methodology, test results, a statement interpreting the test results withtest results, a statement interpreting the test results withclinical and genetic counseling indications and the signature of the clinical and genetic counseling indications and the signature of the laboratorylaboratorydirector or other authorized individual. The final report should be easy todirector or other authorized individual. The final report should be easy tointerpret and should include an appropriate summary of interpret and should include an appropriate summary of the methods, probesthe methods, probesand endonucleases used, the loci or mutations tested, the objective findingsand endonucleases used, the loci or mutations tested, the objective findingsand a clinical interpretation in and a clinical interpretation in an easytointerpret format (JAHCO, 1996).an easytointerpret format (JAHCO, 1996).

The final report should be reviewed and signed by the director or a qualifiedThe final report should be reviewed and signed by the director or a qualifieddesignee if there is a subjective or an designee if there is a subjective or an interpretive component to the test.interpretive component to the test.When diagnostic reports are generated by computer or telecommunicationsWhen diagnostic reports are generated by computer or telecommunicationsequipment the actual signature equipment the actual signature or initials of the director need not appear onor initials of the director need not appear onthe report. Nevertheless, the laboratory must have a procedure that ensuresthe report. Nevertheless, the laboratory must have a procedure that ensures

and and documents that the report has been reviewed and approved prior to itsdocuments that the report has been reviewed and approved prior to itsrelease.release.

4.1 Molecular Inherited Disease Testing4.1 Molecular Inherited Disease Testing

In view of the recognized risks of genetic discrimination and stigmatization,In view of the recognized risks of genetic discrimination and stigmatization,confidentiality of molecular genetic test results is a confidentiality of molecular genetic test results is a critical consideration.critical consideration.Results should be communicated only to the referring physician, geneticResults should be communicated only to the referring physician, geneticcounselor or in certain cases, the patient. counselor or in certain cases, the patient. Non-confidential media (e.g., fax)Non-confidential media (e.g., fax)should be used with caution. Some patients, aware of the insurability risks willshould be used with caution. Some patients, aware of the insurability risks willchoose to pay for choose to pay for testing out of their own pocket and request that the resultstesting out of their own pocket and request that the resultsnot be recorded in their medical record; such requests should be honored bynot be recorded in their medical record; such requests should be honored bythe laboratory to the extent allowable under applicable laws. Under nothe laboratory to the extent allowable under applicable laws. Under nocircumstances should results be provided to outside parties, such circumstances should results be provided to outside parties, such asasemployers, insurers or other family members without the patient's expressemployers, insurers or other family members without the patient's expressconsent. Laboratory workers should even use caution consent. Laboratory workers should even use caution when publishing orwhen publishing orpublicly presenting the results of such studies, as some family members havepublicly presenting the results of such studies, as some family members haverecognized their own pedigrees in recognized their own pedigrees in published material and thereby derivedpublished material and thereby derivedotherwise confidential information (Holtzman and Watson, 1988).otherwise confidential information (Holtzman and Watson, 1988).

Reports of genetic testing of complex disease genes with multiple possibleReports of genetic testing of complex disease genes with multiple possiblemutations, the report should include (where mutations, the report should include (where appropriate) an estimate ofappropriate) an estimate ofresidual risk of being a carrier for one of the mutations not tested for. Theresidual risk of being a carrier for one of the mutations not tested for. Thereport should include a discussion of report should include a discussion of the limitations of the methods/tests andthe limitations of the methods/tests andthe clinical implications of the detected mutation (or negative result) forthe clinical implications of the detected mutation (or negative result) forcomplex disorders complex disorders with regard to recessive or dominant inheritance, recurrencewith regard to recessive or dominant inheritance, recurrencerisk, penetrance, severity and other aspects of genotypephenotype risk, penetrance, severity and other aspects of genotypephenotype correlation.correlation.The report should also include an estimate of the risk of false negatives andThe report should also include an estimate of the risk of false negatives andfalse positives arising from recombination between false positives arising from recombination between the linked probe(s) and thethe linked probe(s) and thedisease gene, when linkage analysis is used.disease gene, when linkage analysis is used.

The report should include a recommendation that appropriate geneticThe report should include a recommendation that appropriate geneticcounseling be utilized to explain the implications of the counseling be utilized to explain the implications of the test results, residualtest results, residualrisks and uncertainties, and the reproductive or medical options it raises to therisks and uncertainties, and the reproductive or medical options it raises to thepatient where appropriate. The patient where appropriate. The genetic councilor will convey sensitivegenetic councilor will convey sensitiveinformation to the patients in an understandable manner.information to the patients in an understandable manner.

4.2 Paternity Testing4.2 Paternity Testing

The report should include the individual paternity index for each geneticThe report should include the individual paternity index for each geneticsystem, the combined paternity index, the probability system, the combined paternity index, the probability of paternity as aof paternity as apercentage, and the prior probabilities used in calculations if there is a failurepercentage, and the prior probabilities used in calculations if there is a failureto exclude (Holtzman and Watson, 1988).to exclude (Holtzman and Watson, 1988).

4.3 4.3 In SituIn Situ Hybridization Hybridization

The interpretive report should include correlation with the morphologicalThe interpretive report should include correlation with the morphologicalfindings. findings. In situIn situ hybridization requires simultaneous hybridization requires simultaneous re-evaluation of there-evaluation of thehistopathology or cytopathology on the actual hybridized slide, sincehistopathology or cytopathology on the actual hybridized slide, sincesectioning or sampling may alter a focal lesion. sectioning or sampling may alter a focal lesion. Consequently, the correlationConsequently, the correlation

between the morphological and molecular data must be described in the reportbetween the morphological and molecular data must be described in the report(Nuovo, 1992).(Nuovo, 1992).

Any report must ensure the confidentiality of the other family members whoseAny report must ensure the confidentiality of the other family members whosestudies were used to provide information to studies were used to provide information to the proband. The format can bethe proband. The format can besuch that one copy is detailed and for the referring genetic expert, while asuch that one copy is detailed and for the referring genetic expert, while acover summary sheet is cover summary sheet is provided for the proband as long as no other familyprovided for the proband as long as no other familymembers' results are revealed.members' results are revealed.

4.4 Investigative Studies4.4 Investigative Studies

A written report must be issued to the referring source and must contain theA written report must be issued to the referring source and must contain thesame information stated previously. However, there same information stated previously. However, there must be a qualifyingmust be a qualifyingstatement clearly indicating that the results are based on an investigationalstatement clearly indicating that the results are based on an investigationalstudy and may not be as accurate as a study and may not be as accurate as a test recognized by the genetictest recognized by the geneticcommunity as an accepted or proven clinical service test.community as an accepted or proven clinical service test.

5. Records5. Records

The laboratory must maintain all patient records of patient data and laboratoryThe laboratory must maintain all patient records of patient data and laboratoryoperations in a manner that permits timely review.operations in a manner that permits timely review.

5.1 Patient Records5.1 Patient Records

All patient testing laboratory records should be accessible and easily retrieved.All patient testing laboratory records should be accessible and easily retrieved.Files should be retrievable by patient name and by Files should be retrievable by patient name and by a second unique identifiera second unique identifier(e.g. laboratory accession number or case number). Files relating to individual(e.g. laboratory accession number or case number). Files relating to individualor or familyfamily studies should be cross-referenced. Records should be maintained in astudies should be cross-referenced. Records should be maintained in amanner that will preserve their confidentiality and integrity and released onlymanner that will preserve their confidentiality and integrity and released onlywith appropriate authorization. The records should be retained for a suitablewith appropriate authorization. The records should be retained for a suitableperiod of time as required by applicable regulations. period of time as required by applicable regulations. Critical records of geneticCritical records of genetictesting are often kept for one generation (i.e. 20 years) (Baer, 1993).testing are often kept for one generation (i.e. 20 years) (Baer, 1993).

5.2 Laboratory Records5.2 Laboratory Records

Records of all components of the internal quality improvement program,Records of all components of the internal quality improvement program,proficiency testing and internal quality control program proficiency testing and internal quality control program should be complete andshould be complete andavailable. Copies of all outdated procedures and the dates for which they wereavailable. Copies of all outdated procedures and the dates for which they werein effect should be retained in effect should be retained for reference. The laboratory records shouldfor reference. The laboratory records shouldinclude sufficient information regarding the individual specimen and assayinclude sufficient information regarding the individual specimen and assayconditions. A log conditions. A log of all stored specimens should be maintained to allow forof all stored specimens should be maintained to allow forprompt retrieval for further testing. Copies of each final report, all records prompt retrieval for further testing. Copies of each final report, all records ofofresults, membranes, autoradiographs, gel photographs and results, membranes, autoradiographs, gel photographs and in situin situ hybridization hybridizationshould also be retained in compliance with should also be retained in compliance with existing laws. All autoradiographs,existing laws. All autoradiographs,gel photographs and gel photographs and in situin situ hybridization slides must be adequately labeled for hybridization slides must be adequately labeled foridentification and identification and adequately cross-referenced in the case records.adequately cross-referenced in the case records.

5.3 Parentage and Forensic Identity Testing5.3 Parentage and Forensic Identity Testing

This includes all technical, legal and administrative records available for reviewThis includes all technical, legal and administrative records available for reviewand use in legal proceedings. The records and use in legal proceedings. The records should reflect an adequate externalshould reflect an adequate external

and internal chain-of-custody. Records pertaining to release of informationand internal chain-of-custody. Records pertaining to release of informationshould be maintained all should be maintained all the time (NCCLS MM1-A, 2000).the time (NCCLS MM1-A, 2000).

6. Reagents6. Reagents

The laboratory has the responsibility for ensuring that all reagents used,The laboratory has the responsibility for ensuring that all reagents used,whether purchased or prepared by the laboratory are whether purchased or prepared by the laboratory are functional. Verification offunctional. Verification ofreagent performance is required and must be documented. Any of severalreagent performance is required and must be documented. Any of severalmethods may be appropriate such as methods may be appropriate such as direct analysis with reference materials,direct analysis with reference materials,parallel testing of old vs. new reagents and checks against routine controls.parallel testing of old vs. new reagents and checks against routine controls.The aim is to check The aim is to check new reagents by an appropriate method before beingnew reagents by an appropriate method before beingplaced in service. Documentation should exist that new reagent lots have placed in service. Documentation should exist that new reagent lots have beenbeenchecked against prior lots or known standards before being placed in service.checked against prior lots or known standards before being placed in service.Reagents intended for use in a manner which do Reagents intended for use in a manner which do not follow manufacturer'snot follow manufacturer'srecommendations requires that validation studies must be performed (CAPrecommendations requires that validation studies must be performed (CAPLaboratory General Checklist, 2001).Laboratory General Checklist, 2001).

All reagents should be properly labeled with content and quantity,All reagents should be properly labeled with content and quantity,concentration, storage requirements, date placed in service concentration, storage requirements, date placed in service and expiration dateand expiration date(all reagents should be used within their indicated expiration date). Upon visual(all reagents should be used within their indicated expiration date). Upon visualinspection all reagents should be inspection all reagents should be in satisfactory condition and should be storedin satisfactory condition and should be storedappropriately as stated by the manufacturers.appropriately as stated by the manufacturers.

Sufficient information should be documented regarding the nature of any probeSufficient information should be documented regarding the nature of any probeor primer used in an assay to permit or primer used in an assay to permit proper interpretation and troubleshootingproper interpretation and troubleshootingof test results such as the type (genomic, cDNA, oligonucleotide or riboprobe)of test results such as the type (genomic, cDNA, oligonucleotide or riboprobe)and origin and origin (human, viral, etc.) of the probe or sequence; the oligonucleotide(human, viral, etc.) of the probe or sequence; the oligonucleotidesequence and complementary sequence or gene region recognized; sequence and complementary sequence or gene region recognized; thetheappropriate restriction enzyme map of the DNA; known polymorphisms; sitesappropriate restriction enzyme map of the DNA; known polymorphisms; sitesresistant to endonuclease digestion and resistant to endonuclease digestion and cross-hybridizing bands; the labelingcross-hybridizing bands; the labelingmethods used and standards for adequacy of hybridization or amplification. Formethods used and standards for adequacy of hybridization or amplification. Forlinkage analysis linkage analysis recombination frequencies and map position must berecombination frequencies and map position must bedocumented. Loci should be designed as defined by the Human Gene Mappingdocumented. Loci should be designed as defined by the Human Gene MappingNomenclature Committee.Nomenclature Committee.

7. Instrument Maintenance7. Instrument Maintenance

The laboratory should have an organized system for monitoring and maintainingThe laboratory should have an organized system for monitoring and maintainingall instruments. Function checks should be all instruments. Function checks should be designed to check the criticaldesigned to check the criticaloperating characteristics to detect drift, instability or malfunction before theoperating characteristics to detect drift, instability or malfunction before theproblem is allowed to affect the problem is allowed to affect the test results. All servicing and repairs must betest results. All servicing and repairs must bedocumented. The procedures and schedules for instrument maintenance mustdocumented. The procedures and schedules for instrument maintenance mustbe be thorough and as frequent as specified by the manufacturer. Since certainthorough and as frequent as specified by the manufacturer. Since certainequipment have no standard frequency or extent of maintenance, equipment have no standard frequency or extent of maintenance, eacheachlaboratory should establish schedules that reasonably reflect the workload andlaboratory should establish schedules that reasonably reflect the workload andspecifications of its equipment. The following specifications of its equipment. The following requirements should be followedrequirements should be followedfor all equipment in the laboratory: i) written standard procedure for set-upfor all equipment in the laboratory: i) written standard procedure for set-upand normal operation of all and normal operation of all instruments in the laboratory; ii) regular scheduleinstruments in the laboratory; ii) regular scheduleor system for checking the critical operation for all instruments in use; iii)or system for checking the critical operation for all instruments in use; iii)function checks function checks should be documented to detect trends or malfunctions; andshould be documented to detect trends or malfunctions; and

iv) instructions should be provided for minor troubleshooting and repair iv) instructions should be provided for minor troubleshooting and repair ofofinstruments (this can be done by providing the manufacturer's service manualinstruments (this can be done by providing the manufacturer's service manualor notes) and records for each instrument repair or notes) and records for each instrument repair history and preventivehistory and preventivemaintenance (these records should be available to and usable by the technicalmaintenance (these records should be available to and usable by the technicalstaff operating the equipment) staff operating the equipment) (CAP Laboratory General Checklist, 2001).(CAP Laboratory General Checklist, 2001).

Electrophoresis EquipmentElectrophoresis Equipment

The electrophoresis apparatus in the laboratory should be clean and properlyThe electrophoresis apparatus in the laboratory should be clean and properlymaintained (electrode and buffer tank intact, maintained (electrode and buffer tank intact, power supply electrodes fitpower supply electrodes fitsnugly and no build up of dried buffer).snugly and no build up of dried buffer).

Power SuppliesPower Supplies

The displayed voltage reading should be checked periodically with a voltmeter,The displayed voltage reading should be checked periodically with a voltmeter,to ensure that it is delivering the correct voltage.to ensure that it is delivering the correct voltage.

Biological Safety CabinetsBiological Safety Cabinets

The biological safety cabinet (or hood) should be certified at least annually toThe biological safety cabinet (or hood) should be certified at least annually toensure that filters are functioning properly and that ensure that filters are functioning properly and that airflow rates meetairflow rates meetspecifications.specifications.

Fume HoodsFume Hoods

Fume hoods (or chemical filtration unit) should be available for any procedureFume hoods (or chemical filtration unit) should be available for any procedureutilizing volatile chemicals and the fume hood should utilizing volatile chemicals and the fume hood should be certified annually tobe certified annually toensure that airflow rates meet specification.ensure that airflow rates meet specification.

PipettesPipettes

The pipettes should be checked for accuracy and reproducibility before beingThe pipettes should be checked for accuracy and reproducibility before beingplaced in service and should also be checked at placed in service and should also be checked at regular intervals thereafter.regular intervals thereafter.

ThermometersThermometers

All thermometers in the laboratory should be checked against an appropriateAll thermometers in the laboratory should be checked against an appropriatethermometric standard device before being placed thermometric standard device before being placed in service.in service.

Temperature Dependent EquipmentTemperature Dependent Equipment

The temperature should be recorded daily (and when used) and the rangesThe temperature should be recorded daily (and when used) and the rangesdefined for the following equipments: water baths, defined for the following equipments: water baths, heating blocks, incubators,heating blocks, incubators,ovens, refrigerators, freezers as well as the individual wells of thermal cyclerovens, refrigerators, freezers as well as the individual wells of thermal cyclershould be checked for should be checked for temperature accuracy and uniformity before beingtemperature accuracy and uniformity before beingplaced in service and periodically thereafter.placed in service and periodically thereafter.

pH MeterspH Meters

There should be written procedures for operation, calibration and functionThere should be written procedures for operation, calibration and functionchecks. High quality buffers (certified assay of checks. High quality buffers (certified assay of content) should be used forcontent) should be used forcalibration of the pH meter.calibration of the pH meter.

CentrifugesCentrifuges

Centrifuges in the laboratory should be cleaned and properly maintained. ThereCentrifuges in the laboratory should be cleaned and properly maintained. Thereshould be a written protocol for maintenance of should be a written protocol for maintenance of all centrifuges and theall centrifuges and theoperating speeds should be checked periodically for the intended use.operating speeds should be checked periodically for the intended use.

Balances and WeightsBalances and Weights

The balances should be cleaned and checked periodically by qualified serviceThe balances should be cleaned and checked periodically by qualified servicepersonnel and all analytical balances should be personnel and all analytical balances should be mounted so that vibrations domounted so that vibrations donot interfere with readings. Results of the periodic accuracy checks should benot interfere with readings. Results of the periodic accuracy checks should berecorded. Standard weights of recorded. Standard weights of the appropriate American Society for Testingthe appropriate American Society for Testingand Materials (ASTM) class should be available for calibration. ASTM Class-Iand Materials (ASTM) class should be available for calibration. ASTM Class-Iweights weights are appropriate for calibrating high precision analytical balances (0.01are appropriate for calibrating high precision analytical balances (0.01to 0.1 mg). ASTM Class-II weights are appropriate for to 0.1 mg). ASTM Class-II weights are appropriate for calibrating high precisioncalibrating high precisionanalytical balances (0.001 to 0.01g). ASTM Class-III weights are appropriateanalytical balances (0.001 to 0.01g). ASTM Class-III weights are appropriatefor calibrating high precision for calibrating high precision analytical balances (0.01 to 0.1g).analytical balances (0.01 to 0.1g).

Volumetric GlasswareVolumetric Glassware

Volumetric glassware should be of a certified accuracy [Class A, NationalVolumetric glassware should be of a certified accuracy [Class A, NationalInstitute of Standards and Technology (NIST) standard Institute of Standards and Technology (NIST) standard or equivalent] or if non-or equivalent] or if non-certified volumetric glassware is used all items are checked for accuracy ofcertified volumetric glassware is used all items are checked for accuracy ofcalibration before being placed calibration before being placed in service.in service.

SpectrophotometersSpectrophotometers

The absorbance and the photometric linearity should be checked periodicallyThe absorbance and the photometric linearity should be checked periodicallywith filters or standards solutions if required by with filters or standards solutions if required by the instrument manufacturer.the instrument manufacturer.The filters (filter photometers) should be checked periodically to ensure theyThe filters (filter photometers) should be checked periodically to ensure theyare in good condition are in good condition (e.g., clean, free of scratches etc). The(e.g., clean, free of scratches etc). Thespectrophotometer wavelength calibration should be checked regularly withspectrophotometer wavelength calibration should be checked regularly withappropriate appropriate solutions, filters or emission line source lamps if so required by thesolutions, filters or emission line source lamps if so required by themanufacturer.manufacturer.

Film Processing/Photographic EquipmentFilm Processing/Photographic Equipment

The film processing (developing) equipment should be routinely serviced,The film processing (developing) equipment should be routinely serviced,repaired and appropriately replenished with reagents, repaired and appropriately replenished with reagents, if maintained by theif maintained by thelaboratory. If the laboratory uses another department's film processinglaboratory. If the laboratory uses another department's film processingequipment, the quality of the equipment, the quality of the autoradiographs produced must be monitored.autoradiographs produced must be monitored.The photographic equipment in the laboratory should be clean and properlyThe photographic equipment in the laboratory should be clean and properlymaintained. Fixed maintained. Fixed camera mountings should be level and secure.camera mountings should be level and secure.

Signal Detection InstrumentsSignal Detection Instruments

Scintillation counters, luminometers, densitometers etc should haveScintillation counters, luminometers, densitometers etc should havebackground levels checked and recorded daily, or with each use background levels checked and recorded daily, or with each use of theof theinstrument if used less than daily. Written criteria for acceptable backgroundinstrument if used less than daily. Written criteria for acceptable backgroundlevels should be included.levels should be included.

8. Personnel8. Personnel

For optimal patient care, only qualified personnel may be involved withFor optimal patient care, only qualified personnel may be involved withmolecular pathology testing.molecular pathology testing.

8.1 Director8.1 Director

A board-certified pathologist, other physician, or a doctoral scientist qualifiedA board-certified pathologist, other physician, or a doctoral scientist qualifiedby training, expertise and experience in molecular by training, expertise and experience in molecular genetics can serve as agenetics can serve as adirector. When a non-pathologist physician or doctoral scientist serves asdirector. When a non-pathologist physician or doctoral scientist serves asdirector, such individuals must have director, such individuals must have documented qualifications. The directordocumented qualifications. The directorshall be qualified to assume the professional, scientific, consultative,shall be qualified to assume the professional, scientific, consultative,organizational, administrative organizational, administrative and educational responsibilities for the scope ofand educational responsibilities for the scope ofthe services provided. In addition, the director shall have sufficient authoritythe services provided. In addition, the director shall have sufficient authorityto to implement and maintain the standards for laboratory accreditation listedimplement and maintain the standards for laboratory accreditation listedelsewhere (CAP Standards for Laboratory Accreditation, 2001).elsewhere (CAP Standards for Laboratory Accreditation, 2001).

8.2 Technical Supervisor8.2 Technical Supervisor

The technical supervisor of the laboratory (or section) may be a pathologist,The technical supervisor of the laboratory (or section) may be a pathologist,certified physician in a specialty other than pathology, or certified physician in a specialty other than pathology, or a doctoral scientista doctoral scientistin a biological science, with specialized training and/or appropriate experiencein a biological science, with specialized training and/or appropriate experiencein molecular genetics. In the case in molecular genetics. In the case of forensic identity testing the technicalof forensic identity testing the technicalsupervisor should have an appropriate degree, training or experience in forensicsupervisor should have an appropriate degree, training or experience in forensicscience science (DHHS, 1988).(DHHS, 1988).

8.3 Technical Staff8.3 Technical Staff

The personnel performing the technical aspects of molecular genetics shouldThe personnel performing the technical aspects of molecular genetics shouldqualify as one of the following: i) staff experienced in qualify as one of the following: i) staff experienced in the field of molecularthe field of moleculargenetics under the direct supervision of a qualified director or supervisor; ii)genetics under the direct supervision of a qualified director or supervisor; ii)medical technologist certified by medical technologist certified by the American Society of Clinical Pathologiststhe American Society of Clinical Pathologists(ASCP) or iii) BS degree holder in biological sciences with appropriate(ASCP) or iii) BS degree holder in biological sciences with appropriateexperience in experience in molecular genetics methods. The person in charge of molecularmolecular genetics methods. The person in charge of moleculargenetics assays should be either a BS degree holder or medical genetics assays should be either a BS degree holder or medical technologisttechnologistcertified by the ASCP with at least four years of experience (at least one ofcertified by the ASCP with at least four years of experience (at least one ofwhich is in molecular genetics methods) under a which is in molecular genetics methods) under a qualified director. Therequalified director. Thereshould be adequate training programs for new technologists in addition toshould be adequate training programs for new technologists in addition tocontinuing medical laboratory continuing medical laboratory education programs (DHHS, 1988).education programs (DHHS, 1988).

9. Laboratory Computer Services9. Laboratory Computer Services

Laboratory computer systems are vital to accurate reporting of patient resultsLaboratory computer systems are vital to accurate reporting of patient resultsand hence to patient care. The following regulations and hence to patient care. The following regulations do NOT apply to smalldo NOT apply to smallprogrammable technical computers, micro computers used solely for wordprogrammable technical computers, micro computers used solely for wordprocessing, spreadsheets or processing, spreadsheets or dedicated microprocessors that are an integraldedicated microprocessors that are an integralpart of an analytic instrument.part of an analytic instrument.

9.1 Environment9.1 Environment

The computer facility and equipment should be kept clean, well maintained andThe computer facility and equipment should be kept clean, well maintained and

adequately ventilated with appropriate adequately ventilated with appropriate temperature and humidity control. Alltemperature and humidity control. Allwires and computer cables should be properly located and/or protected fromwires and computer cables should be properly located and/or protected fromtraffic areas. Computer traffic areas. Computer components and storage areas must be readilycomponents and storage areas must be readilyaccessible to fire-fighting equipment. The computer system must beaccessible to fire-fighting equipment. The computer system must beadequately protected adequately protected against unexpected power interruptions and surges usingagainst unexpected power interruptions and surges usingan Uninterruptible Power System (UPS) or similar device (NCCLS GP19-A,an Uninterruptible Power System (UPS) or similar device (NCCLS GP19-A,1997).1997).

9.2 Personnel and Security9.2 Personnel and Security

Procedure manuals that are written and reviewed annually are necessary. TheProcedure manuals that are written and reviewed annually are necessary. Themanual should have written procedures for the manual should have written procedures for the following: preservation of datafollowing: preservation of dataand equipment in case of an unexpected destructive event and equipment in case of an unexpected destructive event ((e.g.e.g., fire, flood),, fire, flood),software and hardware software and hardware failure, security codes which define levels of securityfailure, security codes which define levels of securityfor those who are authorized to enter, access or change of patient resultsfor those who are authorized to enter, access or change of patient results(information sent over a public domain, such as the Internet is considered in(information sent over a public domain, such as the Internet is considered inthe public domain which is potentially accessible to all parties on the public domain which is potentially accessible to all parties on that networkthat networkand a system, such as "fire walls" and data encryption schemes should be inand a system, such as "fire walls" and data encryption schemes should be inplace) and documentation of training for place) and documentation of training for all personnel.all personnel.

9.3 Data Entry, Reports and Errors9.3 Data Entry, Reports and Errors

There should be a written system to document errors in There should be a written system to document errors in testtest reporting andreporting andtransmission of patient results. A timely system for transmission of patient results. A timely system for error correction that iserror correction that isconvenient to use must be defined. Any error correction must be indicated onconvenient to use must be defined. Any error correction must be indicated onthe test report with both the original the test report with both the original and revised results reported and theand revised results reported and theoperator indicated. Patient data on reports and video displays must beoperator indicated. Patient data on reports and video displays must beperiodically compared periodically compared with original input data to detect errors in datawith original input data to detect errors in datatransmission, storage or processing. The laboratory director must approve attransmission, storage or processing. The laboratory director must approve atleast least annually a review of the content and format of the laboratory patientannually a review of the content and format of the laboratory patientreports to ensure that they effectively communicate laboratory results reports to ensure that they effectively communicate laboratory results andandthat they meet the needs of the medical staff. Reports that display correctedthat they meet the needs of the medical staff. Reports that display correctedresults must clearly indicate that the new results results must clearly indicate that the new results are replacing a previouslyare replacing a previouslyreported incorrect result. This applies to all paper reports, as well as data thatreported incorrect result. This applies to all paper reports, as well as data thatare displayed on video are displayed on video terminals or other systems receiving patient data. Whenterminals or other systems receiving patient data. Whena revised patient report is issued, both the original and revised results must bea revised patient report is issued, both the original and revised results must beretained for at least 2 years. It is considered inappropriate to list only the lastretained for at least 2 years. It is considered inappropriate to list only the lastcorrection made, as the clinician may have made a clinical correction made, as the clinician may have made a clinical decision based upondecision based uponerroneous data rather than the true result. All corrections should be shown inerroneous data rather than the true result. All corrections should be shown inthe patient report. In addition, there must the patient report. In addition, there must be an audit mechanism, which allowsbe an audit mechanism, which allowsthe laboratory to identify all individuals who have entered or modified patientthe laboratory to identify all individuals who have entered or modified patientdata, control files data, control files or computer programs.or computer programs.

9.4 Data Storage and Retrieval9.4 Data Storage and Retrieval

Stored patient result data and archival information must be easily and readilyStored patient result data and archival information must be easily and readilyreviewable within a time frame (less than 4 reviewable within a time frame (less than 4 hours) consistent with patient carehours) consistent with patient careneeds. The computer must be able to reproduce archived test resultsneeds. The computer must be able to reproduce archived test resultscompletely, which include completely, which include the reference range originally given for that test,the reference range originally given for that test,and any flags, footnotes, or interpretive comments that were attached to thatand any flags, footnotes, or interpretive comments that were attached to that

result. result. The tracing of results back to the original instrument on which the testThe tracing of results back to the original instrument on which the testwas performed is important for both quality assurance as well was performed is important for both quality assurance as well as qualityas qualitycontrol. Data storage media such as tapes, disks, etc., must be properlycontrol. Data storage media such as tapes, disks, etc., must be properlylabeled, stored and protected from damage or labeled, stored and protected from damage or unauthorized use. A mechanismunauthorized use. A mechanismmust be in place to minimize or prevent loss of patient information in case ofmust be in place to minimize or prevent loss of patient information in case ofhardware or software failure.hardware or software failure.

9.5 Maintenance9.5 Maintenance

A schedule of maintenance procedures and training for employee should beA schedule of maintenance procedures and training for employee should bedevised. A system to monitor computer resources and devised. A system to monitor computer resources and test system integritytest system integrityshould be in place. Testing should be done after any modifications and resultsshould be in place. Testing should be done after any modifications and resultsdocumented. Approval of new documented. Approval of new programs and changes have to be documentedprograms and changes have to be documentedby the medical director. Policies and procedures must be in effect to minimizeby the medical director. Policies and procedures must be in effect to minimizedowntime and downtime and that any downtime or backup procedures must be documented.that any downtime or backup procedures must be documented.Written contingency plans must be developed to handle services in Written contingency plans must be developed to handle services in the eventthe eventof a computer system failure. Emergency services for both computer hardwareof a computer system failure. Emergency services for both computer hardwareand software must be improved. Records must and software must be improved. Records must be maintained to documentbe maintained to documentregular maintenance and allow operators to trace any work done on theregular maintenance and allow operators to trace any work done on thecomputer system.computer system.

10. Proficiency Testing10. Proficiency Testing

Proficiency testing involves the performance of test procedures on commonProficiency testing involves the performance of test procedures on commonsamples by multiple participant laboratories. samples by multiple participant laboratories. Laboratories must participateLaboratories must participateregularly in a CAP approved proficiency testing program for each patientregularly in a CAP approved proficiency testing program for each patientreportable analyte whenever an reportable analyte whenever an appropriate program is available. Eachappropriate program is available. Eachseparately accredited laboratory must be enrolled in such a program under itsseparately accredited laboratory must be enrolled in such a program under itsown CAP number. It own CAP number. It is preferable that the laboratory has a performance historyis preferable that the laboratory has a performance historyof one or two shipments of proficiency testing before an initial inspection. of one or two shipments of proficiency testing before an initial inspection. IfIfproficiency testing for an analyte is not commercially available, is not formallyproficiency testing for an analyte is not commercially available, is not formallygraded, or is not compatible with all methods, graded, or is not compatible with all methods, the laboratory is still required tothe laboratory is still required toperform some type of external, alternative or comparable testing at least everyperform some type of external, alternative or comparable testing at least everysix months. This may six months. This may be accomplished through blind testing of specimens withbe accomplished through blind testing of specimens withknown results, exchange of specimens with other laboratories, or otherknown results, exchange of specimens with other laboratories, or otherequivalent systems specifically recommended and approved by the laboratoryequivalent systems specifically recommended and approved by the laboratorydirector (NCCLS GP27-A).director (NCCLS GP27-A).

For molecular genetics testing the CAP currently runs the Molecular GeneticsFor molecular genetics testing the CAP currently runs the Molecular GeneticsSurvey program. This program was established Survey program. This program was established with the American College ofwith the American College ofMedical Genetics. The Survey consists of two shipments per year, which areMedical Genetics. The Survey consists of two shipments per year, which aresent to participating sent to participating laboratories. The shipments comes as ethanol fixed celllaboratories. The shipments comes as ethanol fixed celllines or extracted DNA and include the following disease samples: Cysticlines or extracted DNA and include the following disease samples: CysticFibrosis, Fibrosis, DMD/Becker, Factor V Leiden, Fragile X Syndrome, Friedreich's Ataxia,DMD/Becker, Factor V Leiden, Fragile X Syndrome, Friedreich's Ataxia,Hemochromatosis, Hemoglobin S/C, Huntington Hemochromatosis, Hemoglobin S/C, Huntington disease,disease,Methylenetetrahydrofolate reductase, Myotonic dystrophy, Prader-Methylenetetrahydrofolate reductase, Myotonic dystrophy, Prader-Willi/Angelman syndrome, Prothrombin, Rh, Spinal muscular Willi/Angelman syndrome, Prothrombin, Rh, Spinal muscular atrophy andatrophy andSpinocerebellar Ataxia. These specimens should be tested in the same mannerSpinocerebellar Ataxia. These specimens should be tested in the same manneras patient's specimens and by the same as patient's specimens and by the same personnel and the results are sent topersonnel and the results are sent to

the CAP office within the specified period of time.the CAP office within the specified period of time.

The survey intended results are then sent to the laboratory. These should beThe survey intended results are then sent to the laboratory. These should beactively reviewed by the laboratory director actively reviewed by the laboratory director and laboratory personnel andand laboratory personnel andcompared to the actual laboratory results. Testing of the survey samplescompared to the actual laboratory results. Testing of the survey samplesshould be rotated among all should be rotated among all technical personnel when possible (Merrick, 2000).technical personnel when possible (Merrick, 2000).

11.11. Procedure ManualProcedure Manual

The laboratory should have a procedure manual, which includes protocols for allThe laboratory should have a procedure manual, which includes protocols for allassays it performs in sufficient detail to assays it performs in sufficient detail to permit qualified laboratory personnelpermit qualified laboratory personnelto perform them consistently and accurately. The procedure manual should beto perform them consistently and accurately. The procedure manual should bewritten in compliance written in compliance with the NCCLS GP2-A3 (NCCLS, 1997) without having towith the NCCLS GP2-A3 (NCCLS, 1997) without having toprecisely copy it. The procedure manual must include: principles of the precisely copy it. The procedure manual must include: principles of the test,test,clinical significance, specimen type, required reagents and equipments, qualityclinical significance, specimen type, required reagents and equipments, qualitycontrol, procedural steps, calculations, reference control, procedural steps, calculations, reference ranges, interpretation andranges, interpretation andprotocol for reporting of results. Also, there must be documentation on DNAprotocol for reporting of results. Also, there must be documentation on DNAprobes and PCR primers, which probes and PCR primers, which include the size, complete or partial nucleotideinclude the size, complete or partial nucleotidesequence, allele frequencies of certain mutations in various ethnic groups,sequence, allele frequencies of certain mutations in various ethnic groups,recombination frequencies, cloning vector, relevant restriction enzymes sites,recombination frequencies, cloning vector, relevant restriction enzymes sites,method of preparation and relevant literature citations.method of preparation and relevant literature citations.

The procedure manual should also be available to all personnel at theThe procedure manual should also be available to all personnel at theworkbench. It should be reviewed at least annually by workbench. It should be reviewed at least annually by the laboratory directorthe laboratory directorand any changes initialed and dated by the director. When a procedure isand any changes initialed and dated by the director. When a procedure isdiscontinued a copy is maintained for discontinued a copy is maintained for at least two years recording initial dateat least two years recording initial dateof use and retirement date (CAP Molecular Pathology Checklist, 2001).of use and retirement date (CAP Molecular Pathology Checklist, 2001).

There should be a written procedure to prevent specimen loss, alterations orThere should be a written procedure to prevent specimen loss, alterations orcontaminations. The procedure manual contaminations. The procedure manual should document that all analystsshould document that all analystsacknowledge the contents (including changes) of procedure manuals relevantacknowledge the contents (including changes) of procedure manuals relevantto the scope of their to the scope of their testing activities.testing activities.

12. Laboratory Safety12. Laboratory Safety

12.112.1 Chemical HazardsChemical Hazards

Many of the procedures used in molecular genetics laboratories involve the useMany of the procedures used in molecular genetics laboratories involve the useof chemicals which are toxic or mutagenic. of chemicals which are toxic or mutagenic. Appropriate precautions are definedAppropriate precautions are definedin the Material Safety Data Sheets (MSDS) for each chemical and should bein the Material Safety Data Sheets (MSDS) for each chemical and should beincluded in a separate file for included in a separate file for all chemicals in use by the laboratory.all chemicals in use by the laboratory. In In addition,addition,the laboratory should have a Chemical Hygiene Plan (CHP) which defines thethe laboratory should have a Chemical Hygiene Plan (CHP) which defines thesafety procedures for all hazardous chemicals which are in use by thesafety procedures for all hazardous chemicals which are in use by thelaboratory and should contain the following elements: i) laboratory and should contain the following elements: i) responsibilities of labresponsibilities of labdirector and supervisors; ii) designation of a qualified chemical hygiene officer;director and supervisors; ii) designation of a qualified chemical hygiene officer;iii) policies for all operations that involve iii) policies for all operations that involve hazardous chemicals; iv) criteria forhazardous chemicals; iv) criteria forthe use of personal protective equipment and control devices; v) criteria forthe use of personal protective equipment and control devices; v) criteria forexposure monitoring exposure monitoring when possible levels are exceeded; vi) provisions forwhen possible levels are exceeded; vi) provisions formedical consultations and examinations; vii) provision for training employees inmedical consultations and examinations; vii) provision for training employees inthe elements of the CHP and documentation that each chemical used in the elements of the CHP and documentation that each chemical used in thethe lablab

been evaluated for carcinogenic potential, been evaluated for carcinogenic potential, reproductive toxicity. Finally, therereproductive toxicity. Finally, thereshould be an annual review and evaluation of the effectiveness of the CHPshould be an annual review and evaluation of the effectiveness of the CHP(NCCLS GP17-A, 1998).(NCCLS GP17-A, 1998).

12.2 Biological Hazards12.2 Biological Hazards

All human blood specimens are to be treated as infectious and handledAll human blood specimens are to be treated as infectious and handledaccording to the standard precautions. The laboratory according to the standard precautions. The laboratory should have ashould have adocumented policy for infection control that complies with the Occupationaldocumented policy for infection control that complies with the OccupationalSafety and Health Administration (OSHA) Safety and Health Administration (OSHA) standards on occupational exposurestandards on occupational exposureto blood-borne pathogens and to the hospital's exposure control plan.to blood-borne pathogens and to the hospital's exposure control plan.Personnel expected to have Personnel expected to have direct contact with body fluids should receivedirect contact with body fluids should receiveeducation on precautionary measures, epidemiology, modes of transmission andeducation on precautionary measures, epidemiology, modes of transmission andprevention prevention of HIV, Hepatitis-B and Hepatitis-C and the application of universalof HIV, Hepatitis-B and Hepatitis-C and the application of universalprecautions to their work practices. For detailed specific precautions to their work practices. For detailed specific precautions forprecautions forpreventing the laboratory transmission of blood-borne infection frompreventing the laboratory transmission of blood-borne infection fromlaboratory instruments and materials; and laboratory instruments and materials; and recommendations for therecommendations for themanagement of blood borne exposure refer to the NCCLS document M29-management of blood borne exposure refer to the NCCLS document M29-Protection of Laboratory Workers from Protection of Laboratory Workers from Instruments Biohazards and InfectiousInstruments Biohazards and InfectiousDisease Transmitted by Blood Body Fluids and Tissue (NCCLS M-29, 1997).Disease Transmitted by Blood Body Fluids and Tissue (NCCLS M-29, 1997).

12.3 Radiation Safety12.3 Radiation Safety

The institute policy for handling radioactive material should be readily availableThe institute policy for handling radioactive material should be readily availableto all members of staff. There should be an to all members of staff. There should be an up-to-date manual for radiationup-to-date manual for radiationsafety, which should include a section on decontamination and handling ofsafety, which should include a section on decontamination and handling ofradioactive waste. Radiation radioactive waste. Radiation survey instruments should be calibrated regularly.survey instruments should be calibrated regularly.

In all areas or rooms where radioactive materials are being used or stored,In all areas or rooms where radioactive materials are being used or stored,there should be a sign to indicate the presence there should be a sign to indicate the presence of radioactive material. Wipeof radioactive material. Wipetest should be carried out for different radiation areas and records for suchtest should be carried out for different radiation areas and records for suchtest are maintained. In test are maintained. In addition, all workbenches and sinks should be surveyedaddition, all workbenches and sinks should be surveyedand if necessary decontaminated each day of use. A policy should be in place,and if necessary decontaminated each day of use. A policy should be in place,which includes procedures for inspections, monitoring of shipment andwhich includes procedures for inspections, monitoring of shipment andnotification in the event of damaged or leaking radionuclide notification in the event of damaged or leaking radionuclide shipment. Inshipment. Inaddition, all shipments should be logged and the amount used and disposedaddition, all shipments should be logged and the amount used and disposedshould be documented. The laboratory should have should be documented. The laboratory should have a radiation safety officera radiation safety officerwho is responsible and actively monitors radiation safety.who is responsible and actively monitors radiation safety.

12.4 Ultraviolet Hazard12.4 Ultraviolet Hazard

Proper eye and face shields are required when viewing eithidium bromide-Proper eye and face shields are required when viewing eithidium bromide-stained DNA in gels on a UV-trans illuminator box. stained DNA in gels on a UV-trans illuminator box. Severe burns and eyeSevere burns and eyedamage can result from even short exposure to the UV lightdamage can result from even short exposure to the UV light

12.5 Electrical Hazards12.5 Electrical Hazards

Electrophoresis power supplies present an increased shock hazard due to theElectrophoresis power supplies present an increased shock hazard due to theproximity of the electrical supply to liquids and to proximity of the electrical supply to liquids and to the operator. All powerthe operator. All powersupplies should be examined for worn power cords or other signs of damagesupplies should be examined for worn power cords or other signs of damage

before use. Sequencing before use. Sequencing gel electrophoresis presents a special shock hazard duegel electrophoresis presents a special shock hazard dueto the extremely high voltage used (1,500 to 2,500 volts). This type ofto the extremely high voltage used (1,500 to 2,500 volts). This type ofelectrophoresis should not be performed if personnel are not present toelectrophoresis should not be performed if personnel are not present tomonitor the equipment. Unsupervised outside personnel, such as monitor the equipment. Unsupervised outside personnel, such as cleaning staff,cleaning staff,should not be allowed in areas where high voltage gels are being run.should not be allowed in areas where high voltage gels are being run.

13. Quality Control13. Quality Control

The laboratory must have, at a minimum, a written program defining theThe laboratory must have, at a minimum, a written program defining thegeneral quality control policies and procedures in use. general quality control policies and procedures in use. The quality controlThe quality controlprogram should be under surveillance by the section supervisor and reviewedprogram should be under surveillance by the section supervisor and reviewedmonthly by the director or designee.monthly by the director or designee.

DNA should be extracted and purified using standard methods and should beDNA should be extracted and purified using standard methods and should bestored in a manner to prevent degradation. stored in a manner to prevent degradation. There should be a procedure toThere should be a procedure todocument recovery rates of DNA extraction procedure. There should be writtendocument recovery rates of DNA extraction procedure. There should be writtenguidelines for guidelines for handling insufficient or low quantity samples as well ashandling insufficient or low quantity samples as well asspecimens, which do not meet quality standards (Otter and Cooper, specimens, which do not meet quality standards (Otter and Cooper, 1999).1999).Control specimens should be tested in the same manner as patient specimensControl specimens should be tested in the same manner as patient specimensand by the same personnel. Tolerance and acceptability and by the same personnel. Tolerance and acceptability limits should belimits should bedefined for all control procedures, control materials and standards. Results fordefined for all control procedures, control materials and standards. Results forcontrols should be verified for controls should be verified for acceptability before reporting of results. Thereacceptability before reporting of results. Theremust be documentation of all corrective actions taken when controls,must be documentation of all corrective actions taken when controls,instruments, temperature, instruments, temperature, etc exceed defined tolerance limits. Records ofetc exceed defined tolerance limits. Records ofcontrols, instrument function and maintenance, temperature etc should becontrols, instrument function and maintenance, temperature etc should bedocumented documented for routine procedures (Husiman, 1994).for routine procedures (Husiman, 1994).

13.1 Controlling False Positive Nucleic Acid Target Amplification Reactions13.1 Controlling False Positive Nucleic Acid Target Amplification Reactions

Extreme care and measures should be taken to avoid false positive resultsExtreme care and measures should be taken to avoid false positive resultswhen dealing with diagnostic nucleic acid when dealing with diagnostic nucleic acid amplification methods. Theseamplification methods. Thesemeasures can include the following:measures can include the following:

Reagents and SolutionsReagents and Solutions

All reagents used in nucleic acid amplification should be prepared, divided intoAll reagents used in nucleic acid amplification should be prepared, divided intoaliquots and stored in an area that is separate from aliquots and stored in an area that is separate from the specimen preparationthe specimen preparationor post-amplification area. Dedicated equipment and supplies should be used.or post-amplification area. Dedicated equipment and supplies should be used.Oligonucleotides should Oligonucleotides should be synthesized and purified in a clean, amplificationbe synthesized and purified in a clean, amplificationproduct-free environment. Once reaction conditions have been optimizedproduct-free environment. Once reaction conditions have been optimizedreagents can be premixed into master mixes. These master mixes can bereagents can be premixed into master mixes. These master mixes can bedivided into aliquots of the volumes required for each reaction run. divided into aliquots of the volumes required for each reaction run. This willThis willminimize the number of samplings and reduce the potential for contamination.minimize the number of samplings and reduce the potential for contamination.The reagent lot number should be recorded so The reagent lot number should be recorded so that if carryover does occur thethat if carryover does occur thesource can be easily identified (Kitchin, source can be easily identified (Kitchin, et alet al., 1990).., 1990).

PipettesPipettes

Separate pipettes should be used for reagent preparation, specimenSeparate pipettes should be used for reagent preparation, specimenpreparation and post-amplification analysis. Pipettes used preparation and post-amplification analysis. Pipettes used for nucleic acidfor nucleic acid

amplification set-up should always be separated from amplified products andamplification set-up should always be separated from amplified products andshould remain in the area in which they should remain in the area in which they are used. Positive displacementare used. Positive displacementpipettes or barrier pipette tips should be used to prevent contamination ofpipettes or barrier pipette tips should be used to prevent contamination ofpipette barrels by aerosols. pipette barrels by aerosols. All pipettes should be cleaned at regular intervals.All pipettes should be cleaned at regular intervals.

GlovesGloves

Disposable gloves should be worn and changed when entering or re-enteringDisposable gloves should be worn and changed when entering or re-enteringthe amplification-preparation area. Disposable the amplification-preparation area. Disposable gloves may be changed betweengloves may be changed betweensamples to prevent cross-contamination between samples.samples to prevent cross-contamination between samples.

Laboratory CoatsLaboratory Coats

Laboratory coats should be dedicated to areas and changed when travelingLaboratory coats should be dedicated to areas and changed when travelingamong reagent preparation, sample preparation among reagent preparation, sample preparation and amplification and detectionand amplification and detectionareas.areas.

Uncapping Reaction TubesUncapping Reaction Tubes

To force any liquid down from the sides it is recommended that the tubes beTo force any liquid down from the sides it is recommended that the tubes besubjected to a quick centrifugation before subjected to a quick centrifugation before uncapping. Tubes should beuncapping. Tubes should beuncapped carefully to prevent aerosolization.uncapped carefully to prevent aerosolization.

Addition of Reaction ComponentsAddition of Reaction Components

Non-sample components (mineral oil, dNTPs, primers, buffer and enzymes)Non-sample components (mineral oil, dNTPs, primers, buffer and enzymes)should be added to the amplification reactions should be added to the amplification reactions before addition of the sample.before addition of the sample.When possible, before proceeding to the next tube, each tube should beWhen possible, before proceeding to the next tube, each tube should becapped after the addition of capped after the addition of the sample.the sample.

Reagent BlankReagent Blank

These controls contain all necessary components of the amplification reactionThese controls contain all necessary components of the amplification reactionexcept the template DNA and should be included in except the template DNA and should be included in each assay.each assay.

Workflow and Laboratory DesignWorkflow and Laboratory Design

Ideally, three physically separate areas of the laboratory should be available forIdeally, three physically separate areas of the laboratory should be available forreagent preparation, specimen preparation reagent preparation, specimen preparation and amplification and productand amplification and productdetection. The reagent preparation area, for those laboratories using onlydetection. The reagent preparation area, for those laboratories using onlycommercially available kits, commercially available kits, is considered to be the site of the manufacture. Inis considered to be the site of the manufacture. Ina laboratory where enzymatic or chemical means of inactivating amplifieda laboratory where enzymatic or chemical means of inactivating amplifiedproducts products are used, the demands for physical separation of pre- and postare used, the demands for physical separation of pre- and postamplification procedures may be somewhat reduced, but good amplification procedures may be somewhat reduced, but good laboratorylaboratorypractice should still be diligently exercised. The flow in the laboratory shouldpractice should still be diligently exercised. The flow in the laboratory shouldbe in a unidirectional workflow from clean to dirty be in a unidirectional workflow from clean to dirty areas. The use of aareas. The use of aunidirectional workflow will reduce the opportunity for contamination to occur.unidirectional workflow will reduce the opportunity for contamination to occur.Color-coded equipment, reagents Color-coded equipment, reagents and supplies may help to ensure that aand supplies may help to ensure that aunidirectional workflow is maintained (NCCLS GP18-A, 1991).unidirectional workflow is maintained (NCCLS GP18-A, 1991).

In the event that laboratory space is not available to separate pre- and post-In the event that laboratory space is not available to separate pre- and post-

amplification a Class-II biological safety amplification a Class-II biological safety cabinet should be used for specimencabinet should be used for specimenpreparation. Class I safety cabinets do not provide protection for materialpreparation. Class I safety cabinets do not provide protection for materialcontained within them. Dead contained within them. Dead air boxes with ultraviolet light attachment canair boxes with ultraviolet light attachment canprovide a clean bench area for specimen preparation in a dedicated provide a clean bench area for specimen preparation in a dedicated specimen-specimen-preparation laboratory.preparation laboratory.

14. Quality Improvement14. Quality Improvement

The quality improvement (QI) program in molecular genetics must be underThe quality improvement (QI) program in molecular genetics must be underactive surveillance by the supervisor with active surveillance by the supervisor with documented review at least weekly.documented review at least weekly.Secondary review should occur at least monthly by the laboratory director orSecondary review should occur at least monthly by the laboratory director ordesignee.designee. The QI program must provide the system design and evaluation ofThe QI program must provide the system design and evaluation ofproper patient identification and preparation; specimen collection,proper patient identification and preparation; specimen collection,identification, identification, preservation,preservation, transportation, processing and accurate resulttransportation, processing and accurate resultreporting. This system must ensure optimum patient specimen and resultreporting. This system must ensure optimum patient specimen and resultintegrity throughout the pretesting, testing, and posttesting processes.integrity throughout the pretesting, testing, and posttesting processes.Opportunities for system improvement are identified based on Opportunities for system improvement are identified based on such evaluationssuch evaluationsand corrective plans are developed and and corrective plans are developed and implemented.implemented. Evidence of active reviewEvidence of active reviewof results of controls, instrument of results of controls, instrument maintenance and function, temperature, etc.maintenance and function, temperature, etc.for routine procedures should be available on all shifts. There must be afor routine procedures should be available on all shifts. There must be awrittenwritten system in operation to detect and correct significant clerical andsystem in operation to detect and correct significant clerical andanalytic errors that could affect patient management. Failed nucleic acidanalytic errors that could affect patient management. Failed nucleic acidisolations should isolations should be recorded, and documentation should be maintained for allbe recorded, and documentation should be maintained for allcorrective action(s) taken. The submitting physician (or requester) should corrective action(s) taken. The submitting physician (or requester) should bebenotified promptly, when a specimen is inadequate or if insufficient nucleic acidnotified promptly, when a specimen is inadequate or if insufficient nucleic acidis isolated. Failed hybridization reactions should is isolated. Failed hybridization reactions should be recorded and the correctivebe recorded and the correctiveaction documented. There should be evidence that the laboratory monitorsaction documented. There should be evidence that the laboratory monitorssample turnaround times sample turnaround times and that they are appropriate for the intendedand that they are appropriate for the intendedpurpose of the test (Hoeltge, 2000). purpose of the test (Hoeltge, 2000). PreliminaryPreliminary reports should be promptlyreports should be promptlygenerated generated when indicated. Discrepancies between preliminary and final reportswhen indicated. Discrepancies between preliminary and final reportsshould be investigated and documented. Discrepancies between should be investigated and documented. Discrepancies between the molecularthe moleculargenetics laboratory's final results, other laboratory findings, and the clinicalgenetics laboratory's final results, other laboratory findings, and the clinicalpresentation should be investigated presentation should be investigated and documented, along with any necessaryand documented, along with any necessarycorrective action. All abnormal prenatal testing results should be confirmedcorrective action. All abnormal prenatal testing results should be confirmedafter birth after birth or termination of pregnancy, where feasible. There should beor termination of pregnancy, where feasible. There should bestatistical records on all molecular genetics laboratory results statistical records on all molecular genetics laboratory results ((e.g.e.g.,,percentages of normal and abnormal findings, allele frequencies, etc). Thesepercentages of normal and abnormal findings, allele frequencies, etc). Theserecords should be maintained and appropriate records should be maintained and appropriate comparativecomparative studies performed.studies performed.These records should be reviewed at regular intervals by the laboratoryThese records should be reviewed at regular intervals by the laboratorydirector or designee and appropriate director or designee and appropriate corrective action taken should be takencorrective action taken should be takenwhen indicated. A log of unusual, difficult, or instructive cases should bewhen indicated. A log of unusual, difficult, or instructive cases should bemaintained.maintained.

The Accreditation FeesThe Accreditation Fees

A deposit to initiate the application process is currently set at 500 US dollars,A deposit to initiate the application process is currently set at 500 US dollars,which goes towards the first year's accreditation fees. which goes towards the first year's accreditation fees. In addition there areIn addition there arefees for Checklist sections dependent on the number of Checklist sections usedfees for Checklist sections dependent on the number of Checklist sections usedto inspect the laboratory, which to inspect the laboratory, which is currently as follows:is currently as follows:

1-4 Checklist sections / Year $ 9501-4 Checklist sections / Year $ 950

5-8 Checklist sections / Year $ 1,5805-8 Checklist sections / Year $ 1,580

9 Checklist sections /Year $ 1,860 plus $ 270 per checklist > 99 Checklist sections /Year $ 1,860 plus $ 270 per checklist > 9

The inspection team expenses such as airline tickets, hotel accommodation andThe inspection team expenses such as airline tickets, hotel accommodation andmeals are paid for by the CAP central office. There meals are paid for by the CAP central office. There are no hidden costs for theare no hidden costs for theaccreditation process. However, it has to be borne in mind that the cost ofaccreditation process. However, it has to be borne in mind that the cost ofparticipating in the molecular participating in the molecular genetics survey (proficiency testing), which isgenetics survey (proficiency testing), which iscurrently $ 1,200, should be added to the overall cost of accreditation.currently $ 1,200, should be added to the overall cost of accreditation.

Granting / Denial of AccreditationGranting / Denial of Accreditation

Accreditation is formally granted to molecular genetics laboratories that:Accreditation is formally granted to molecular genetics laboratories that:Successfully meet the standards for laboratory Successfully meet the standards for laboratory accreditation set forth by theaccreditation set forth by theCAP, correct and document correction of all deficiencies, cited duringCAP, correct and document correction of all deficiencies, cited duringinspection, within the specified time inspection, within the specified time frames, Successfully participate in aframes, Successfully participate in amolecular genetics survey for all tested analytes, participate in mid-cycle self-molecular genetics survey for all tested analytes, participate in mid-cycle self-evaluation processes evaluation processes and resolve all issues and questions to the satisfaction ofand resolve all issues and questions to the satisfaction ofCAP technical associates and regional commissioners. Laboratory staff isCAP technical associates and regional commissioners. Laboratory staff isrequired to respond to all communication concerning requests for additionalrequired to respond to all communication concerning requests for additionalinformation by CAP, personal consultation with laboratory information by CAP, personal consultation with laboratory directors and re-directors and re-inspection of specific laboratories or laboratory areas (CAP Standards forinspection of specific laboratories or laboratory areas (CAP Standards forAccreditation, 2000). Denial or revocation of Accreditation, 2000). Denial or revocation of accreditation is possible whenaccreditation is possible whenthe laboratory does not respond to the deficiencies cited at the on-sitethe laboratory does not respond to the deficiencies cited at the on-siteinspection, fails to correct and document inspection, fails to correct and document major deficiencies, fails to meet themajor deficiencies, fails to meet theCAP standards for laboratory accreditation or does not participate in a self-CAP standards for laboratory accreditation or does not participate in a self-evaluation. Denial or evaluation. Denial or revocation requires a vote of the entire commission onrevocation requires a vote of the entire commission onlaboratory accreditation or a vote of the executive committee of thelaboratory accreditation or a vote of the executive committee of thecommission. commission. The commission or executive committee is presented with factsThe commission or executive committee is presented with factssurrounding the inspection, after which a vote is taken. Denial is followed surrounding the inspection, after which a vote is taken. Denial is followed by aby acertified letter to the laboratory director, effective immediately and reportedcertified letter to the laboratory director, effective immediately and reportedto the appropriate oversight agencies. The laboratory to the appropriate oversight agencies. The laboratory may appeal the decisionmay appeal the decisionwithin 60 days of notice. Documentation of compliance with all standards mustwithin 60 days of notice. Documentation of compliance with all standards mustbe submitted to the be submitted to the commission. The director may be invited to present thecommission. The director may be invited to present theinformation at a commission meeting if facts not previously reviewed areinformation at a commission meeting if facts not previously reviewed areprovided that provided that may affect the decision. Should the commission adhere to itsmay affect the decision. Should the commission adhere to itsoriginal decision to revoke or deny accreditation, the laboratory may appeal original decision to revoke or deny accreditation, the laboratory may appeal totothe college board of governors. Three members will review the documentation,the college board of governors. Three members will review the documentation,and, if the appeal is considered valid, will refer the and, if the appeal is considered valid, will refer the final decision to the entirefinal decision to the entireboard of governors (Merrick, 2000).board of governors (Merrick, 2000).

Evaluation and FeedbackEvaluation and Feedback

To ensure that the accreditation program meets the members needs, anTo ensure that the accreditation program meets the members needs, anevaluation is included as part of the process. Each facility evaluation is included as part of the process. Each facility is requested tois requested tocomplete a post assessment questionnaire to provide feedback on thecomplete a post assessment questionnaire to provide feedback on theaccreditation process. By doing this, the CAP accreditation process. By doing this, the CAP can ensure that the laboratorycan ensure that the laboratoryaccreditation program is meeting the set goals and that modifications andaccreditation program is meeting the set goals and that modifications and

improvements are implemented improvements are implemented as necessary.as necessary.

ConclusionConclusion

This review has brought together, in one document, all the up-to-dateThis review has brought together, in one document, all the up-to-dateinformation concerning requirements for CAP accreditation information concerning requirements for CAP accreditation in molecularin moleculargenetics laboratories. The CAP accreditation is dependent upon successfulgenetics laboratories. The CAP accreditation is dependent upon successfulperformance in the molecular genetics performance in the molecular genetics survey and passing the laboratorysurvey and passing the laboratoryinspection. The inspection is carried out by practicing laboratorians who haveinspection. The inspection is carried out by practicing laboratorians who haveexperience in the field experience in the field of molecular genetics. They examine all activities carriedof molecular genetics. They examine all activities carriedout in the laboratory ranging from specimen receipt to reporting of results, out in the laboratory ranging from specimen receipt to reporting of results, andandall aspects related to laboratory safety, equipment and computer databases.all aspects related to laboratory safety, equipment and computer databases.Once all requirements for laboratory accreditation Once all requirements for laboratory accreditation are met, the laboratory willare met, the laboratory willbe accredited for a two-year period. Although the accreditation requirementsbe accredited for a two-year period. Although the accreditation requirementsmentioned in this review are mentioned in this review are those for CAP, readers who are looking forthose for CAP, readers who are looking foraccreditation by other agencies or simply looking for a document summarizingaccreditation by other agencies or simply looking for a document summarizinggood good laboratory practices in the field of molecular genetics may find thislaboratory practices in the field of molecular genetics may find thisreview helpful.review helpful.

ReferencesReferences

Anonymous. Using Proficiency Testing (PT) to improve the Clinical Laboratory.Anonymous. Using Proficiency Testing (PT) to improve the Clinical Laboratory.Approved Guideline. 1997. National Committee Approved Guideline. 1997. National Committee for Clinical Laboratoryfor Clinical LaboratoryStandards Approved Guidelines Standards Approved Guidelines ((GP27-AGP27-A), Wayne, PA.), Wayne, PA.

Abu-Amero, K.K., Al-Ahdal, M.N., and Aboul-Enein, H.Y. 2001. An Overview onAbu-Amero, K.K., Al-Ahdal, M.N., and Aboul-Enein, H.Y. 2001. An Overview onLaboratory Accreditation Program of the College Laboratory Accreditation Program of the College of American Pathologists.of American Pathologists.Accred. Qual. Assur. (In Press).Accred. Qual. Assur. (In Press).

Accreditation Manual for Pathology and Clinical Laboratory Services. 1996. By:Accreditation Manual for Pathology and Clinical Laboratory Services. 1996. By:The Joint Commission on Accreditation of Health The Joint Commission on Accreditation of Health Care OrganisationsCare Organisations. . Chicago:Chicago:IL.IL.

Baer, D.M. 1993. Patient records: what to save, how to save it, how long toBaer, D.M. 1993. Patient records: what to save, how to save it, how long tosave it. Med Lab Observ. 25: 22-27.save it. Med Lab Observ. 25: 22-27.

Brown, T.A. 1991a. DNA Amplification by the Polymerase Chain Reaction. In:Brown, T.A. 1991a. DNA Amplification by the Polymerase Chain Reaction. In:Essential Molecular Biology (Practical Approach). Essential Molecular Biology (Practical Approach). Oxford University Press,Oxford University Press,Oxford. p. 185-205.Oxford. p. 185-205.

Brown, T.A. 1991b. Purification of DNA. In: Essential Molecular BiologyBrown, T.A. 1991b. Purification of DNA. In: Essential Molecular Biology(Practical (Practical ApproachApproach).). Oxford University Press, Oxford. p. Oxford University Press, Oxford. p. 47-68.47-68.

Brown, T.A. 1991c.Brown, T.A. 1991c. Restriction Enzyme Digestion. In: Essential MolecularRestriction Enzyme Digestion. In: Essential MolecularBiology (Practical Approach). Oxford University Press, Biology (Practical Approach). Oxford University Press, Oxford. p. 144-150.Oxford. p. 144-150.

Brown, T.A. 1993.Brown, T.A. 1993. Southern Blotting. In: Current Protocols in Molecular Biology.Southern Blotting. In: Current Protocols in Molecular Biology.New York: Wiley Interscience.New York: Wiley Interscience.

Brown, T.A. 1996. Measurement of DNA Concentration. In: Gene Cloning.Brown, T.A. 1996. Measurement of DNA Concentration. In: Gene Cloning.Chapman and Hall, London. p. 34.Chapman and Hall, London. p. 34.

Clinical Laboratories Improvement Clinical Laboratories Improvement Amendments.Amendments. 1988. In: Code of Federal1988. In: Code of FederalRegister. US Department of Health and Human Register. US Department of Health and Human Services. Washington, DC. TitleServices. Washington, DC. Title4242, , Part 493.Part 493.

Clinical Laboratory Safety. 1998. In: National Committee for Clinical LaboratoryClinical Laboratory Safety. 1998. In: National Committee for Clinical LaboratoryStandards Approved Guideline (GP17-A), Wayne, PA.Standards Approved Guideline (GP17-A), Wayne, PA.

Clinical Laboratory Technical Procedure Manuals. 1997. National Committee forClinical Laboratory Technical Procedure Manuals. 1997. National Committee forClinical Laboratory Standards Approved Clinical Laboratory Standards Approved Guideline (GP2-A3), Wayne, PA.Guideline (GP2-A3), Wayne, PA.

Criteria for the Clinical Laboratory Director. 1993. In: Policies and GuidelinesCriteria for the Clinical Laboratory Director. 1993. In: Policies and GuidelinesManual (Appendix O), College of American Manual (Appendix O), College of American Pathologists. Northfield, IL, USA.Pathologists. Northfield, IL, USA.

Erlich, H.A. 1999. Principles and Applications of the Polymerase Chain Reaction.Erlich, H.A. 1999. Principles and Applications of the Polymerase Chain Reaction.Rev Rev Immunogenet.Immunogenet. 1: 127-134.1: 127-134.

Evacuated tubes for blood specimen collection. 1991. National Committee forEvacuated tubes for blood specimen collection. 1991. National Committee forClinical Laboratory Standards Approved Standards Clinical Laboratory Standards Approved Standards (H1-A3), Wayne, PA.(H1-A3), Wayne, PA.

Fischer, S.G. and Lerman, L.S. 1983. DNA fragments differing by single base-Fischer, S.G. and Lerman, L.S. 1983. DNA fragments differing by single base-pair substitutions are separated in denaturing pair substitutions are separated in denaturing gradient gels: correspondencegradient gels: correspondencewith melting theory. Proc Natl Acad Sci. 80: 1579-1584.with melting theory. Proc Natl Acad Sci. 80: 1579-1584.

Glavic, D. and Dean, M. 1995. Applications of heteroduplex analysis forGlavic, D. and Dean, M. 1995. Applications of heteroduplex analysis formutation detection in disease genes. Hum Mutat. 6: 281-287.mutation detection in disease genes. Hum Mutat. 6: 281-287.

Hamlin, W.B. and Duckworth, J.K. 1997. The College of American PathologistsHamlin, W.B. and Duckworth, J.K. 1997. The College of American Pathologists(1946-1996) Laboratory Accreditation (1946-1996) Laboratory Accreditation (Historical Perspective). Arch Pathol(Historical Perspective). Arch PatholLab Med. 121: 745-753.Lab Med. 121: 745-753.

Hoeltge GA. 2000. Your Quality Improvement Plan. Presentation downloadedHoeltge GA. 2000. Your Quality Improvement Plan. Presentation downloadedfrom the College of American Pathologists web site from the College of American Pathologists web site at www.cap.org.at www.cap.org.

Holtzman, N.A. and Watson, M.S. 1988. Promoting Safe and Effective GeneticHoltzman, N.A. and Watson, M.S. 1988. Promoting Safe and Effective GeneticTesting in the United States. Final Report of the Testing in the United States. Final Report of the Task Force on Genetic Testing.Task Force on Genetic Testing.The John Hopkins University Press, Baltimore, MD.The John Hopkins University Press, Baltimore, MD.

Husiman, W. 1994. Quality System in the Medical Laboratory: The Role of aHusiman, W. 1994. Quality System in the Medical Laboratory: The Role of aquality Manual. Annales de Biologie Clinique. 52: 457-461.quality Manual. Annales de Biologie Clinique. 52: 457-461.

Kitchin, P.A., Szotyori, Z., Fromholc, C. and Almond, N. 1990. Avoidance ofKitchin, P.A., Szotyori, Z., Fromholc, C. and Almond, N. 1990. Avoidance offalse positives. Nature. 344: 201-203.false positives. Nature. 344: 201-203.

Laboratory Accreditation Program. 2000. In: Standards for LaboratoryLaboratory Accreditation Program. 2000. In: Standards for LaboratoryAccreditation. College of American Pathologists, Northfield, IL.Accreditation. College of American Pathologists, Northfield, IL.

Laboratory Design. 1991. In: National Committee for Clinical LaboratoryLaboratory Design. 1991. In: National Committee for Clinical LaboratoryStandards, Approved Guideline (GP18-A), Wayne, PA.Standards, Approved Guideline (GP18-A), Wayne, PA.

Laboratory General Inspection Checklist. 2001. In: Laboratory AccreditationLaboratory General Inspection Checklist. 2001. In: Laboratory AccreditationProgram, College of American Pathologists, Northfield, IL.Program, College of American Pathologists, Northfield, IL.

Laboratory Instruments and Data Management Systems. 1997. Design ofLaboratory Instruments and Data Management Systems. 1997. Design ofSoftware User Interfaces and End-User Software Software User Interfaces and End-User Software Systems Validation, OperationSystems Validation, Operationand Monitoring. National Committee for Clinical Laboratory Standards Approvedand Monitoring. National Committee for Clinical Laboratory Standards ApprovedGuideline (GP19-A), Guideline (GP19-A), Wayne, PA.Wayne, PA.

Maniatis, T., Fritsch, E.F. and Sambrook, J. 1989. SDS - polyacrylamide gelManiatis, T., Fritsch, E.F. and Sambrook, J. 1989. SDS - polyacrylamide gelelectrophoresis of proteins. In: Molecular Cloning: electrophoresis of proteins. In: Molecular Cloning: A laboratory manual. ColdA laboratory manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York.Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Maniatis, T., Fritsch, E.F. and Sambrook, J. 1989. Sequencing techniques andManiatis, T., Fritsch, E.F. and Sambrook, J. 1989. Sequencing techniques andstrategies. In: Molecular Cloning: A laboratory strategies. In: Molecular Cloning: A laboratory manual. Cold Spring Harbormanual. Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York.Laboratory Press, Cold Spring Harbor, New York.

Merrick, T. 2000. Laboratory Accreditation Program. Merrick, T. 2000. Laboratory Accreditation Program. InIn: : LaboratoryLaboratoryAccreditation Manual. College of American Pathologists, Accreditation Manual. College of American Pathologists, Northfield, IL.Northfield, IL.

Molecular Diagnostic Methods for Genetic Diseases.2000. National CommitteeMolecular Diagnostic Methods for Genetic Diseases.2000. National Committeefor Clinical Laboratory Standards. Approved for Clinical Laboratory Standards. Approved Guidelines (MM1-A), Wayne, PA.Guidelines (MM1-A), Wayne, PA.

Molecular Pathology Inspection Checklist. 2001. In: Laboratory AccreditationMolecular Pathology Inspection Checklist. 2001. In: Laboratory AccreditationProgram, College of American Pathologists, Program, College of American Pathologists, Northfield, IL.Northfield, IL.

Nuovo, G.J. 1992. PCR Nuovo, G.J. 1992. PCR in situin situ Hybridization. In: Protocols and Applications. Hybridization. In: Protocols and Applications.Raven Press, New York.Raven Press, New York.

Orita, M.Orita, M. 1989. Detection of polymorphisms of human DNA by gel1989. Detection of polymorphisms of human DNA by gelelectrophoresis as single-strand conformation polymorphisms. electrophoresis as single-strand conformation polymorphisms. Proc Natl AcadProc Natl AcadSci. 86: 2766-2770.Sci. 86: 2766-2770.

Ott, J. 1991. Analysis of Human Genetic Linkage. The John Hopkins UniversityOtt, J. 1991. Analysis of Human Genetic Linkage. The John Hopkins UniversityPress, Baltimore, MD.Press, Baltimore, MD.

Otter, J. and Cooper, E.S. 1999. What do Accreditation Organizations Expect?.Otter, J. and Cooper, E.S. 1999. What do Accreditation Organizations Expect?.Arch Pathol Lab Med. 123: 468-471.Arch Pathol Lab Med. 123: 468-471.

Protection of Laboratory Workers from Instrument Biohazards and InfectiousProtection of Laboratory Workers from Instrument Biohazards and InfectiousDiseases transmitted by Blood, Body fluids and Diseases transmitted by Blood, Body fluids and Tissues. 1997. NationalTissues. 1997. NationalCommittee for Clinical Laboratory Standards Approved Guideline (M-29),Committee for Clinical Laboratory Standards Approved Guideline (M-29),Wayne, PA.Wayne, PA.

Rosenstraus, M., Wang, Z., Chang, S.Y. 1998. An Internal Control for RoutineRosenstraus, M., Wang, Z., Chang, S.Y. 1998. An Internal Control for RoutineDiagnostic PCR: Design, Properties and Effect Diagnostic PCR: Design, Properties and Effect on Clinical Performance. J Clinon Clinical Performance. J ClinMicrobiol. 36: 191-197.Microbiol. 36: 191-197.

Standards and Guidelines for Clinical Genetics Laboratories. 1999. AmericanStandards and Guidelines for Clinical Genetics Laboratories. 1999. AmericanCollege of Medical Genetics (ACMG). USA.College of Medical Genetics (ACMG). USA.

Tsongalis, G.J., Wu AH, Silver, H Tsongalis, G.J., Wu AH, Silver, H et alet al. 1999. Applications of Forensic Identity. 1999. Applications of Forensic IdentityTesting in Clinical Laboratory. Am J Clin Pathol. 112: Testing in Clinical Laboratory. Am J Clin Pathol. 112: 93-103.93-103.

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