ASHI Governance 

Standards for Histocompatibility Testing
(4/98) Copyright © 1995 - 2002 American Society for Histocompatibility and Immunogenetics. All rights reserved.

Section P - Nucleic Acid Analysis

The nucleic acid analysis standards apply to histocompatibility testing.

P1.000 Restriction Fragment Length Polymorphism (RFLP)

P1.100 Restriction endonucleases

P1.110 Enzymes must be stored and utilized under conditions recommended by the manufacturer (i.e. storage temperature, test temperature, buffer) to ensure proper DNA digestion.

P1.120 It should be documented that each lot of enzyme produces human DNA polymorphism of known sizes prior to analysis of results.

P1.130 When DNA is digested for analysis, human DNA which will produce polymorphism of known sizes must also be digested to ensure complete endonuclease digestion.

P1.200 Probes

P1.210 Each DNA probe utilized should be validated by family studies demonstrating Mendelian inheritance of the polymorphism detected and by extensive population studies.

P1.220 The probe should be used in the form as reported in the scientific literature and as was used to determine the inheritance pattern and population distribution of the polymorphism.

P1.300 DNA Extraction

P1.310 DNA should be purified by a standard method that has been reported in the scientific literature and validated in the laboratory.

P1.320 If the DNA is not used immediately after purification, suitable methods of storage should be available that would protect the integrity of the material.

P1.330 DNA must be intact and not degraded.

P1.400 Electrophoresis

P1.410 Size markers of known sequences that give discrete electrophoretic bands that span and flank the entire range of the DNA system being tested must be included in the electrophoretic run. The known human control DNA used to determine that complete endonuclease digestion was achieved, must also be included in each electrophoretic run as a control.

P1.420 Equal amounts (mg/ml) of DNA must be loaded per lane.

P1.430 A photograph of the ethidium bromide pattern resulting from the electrophoretic separation should be kept for each run.

P1.500 Prehybridization, hybridization, autoradiography

P1.510 Prehybridization, hybridization, autoradiography must be carried out under empirically determined conditions of concentration, temperature and salt concentration which are determined by the nature of the probe.

P1.520 Stringency conditions should be selected to minimize the possibility of cross-hybridization.

P1.530 Probes should be labeled by a method appropriate for the probe in use. Nick translation, hexamer priming, end labeling or avidin biotin may be appropriate.

P1.540 Each probe used should give a signal adequate to detect a single copy gene. Whenever possible, locus-specific probes should be used.

P1.550 Re-probing of the same membrane should be performed only after complete stripping of the first probe.

P1.600 Analysis

P1.610 Only autoradiographs or membranes that reveal the appropriate patterns of the human control DNA and size markers should be analyzed.

P1.620 Each autoradiograph or membrane should be read independently by two or more individuals.

P1.630 The laboratory report for each fragment detected should specify the probe, restriction endonuclease used, fragment size (k.b.) and the chromosomal location as defined by the International Human Gene Mapping Workshop.

P2.000 Amplification-based Typing

P2.1000 Amplification

P2.1100 Laboratory Design.

Use of physical and/or biochemical barriers to prevent DNA contamination (carry-over) is required. Pre-amplification procedures must be performed in a dedicated work area that excludes amplified DNA that has the potential to serve as a template for amplification in the HLA typing assays (e.g., PCR product, plasmids containing HLA genes). Physical separation and restricted traffic flow is recommended. Use of a static air hood or a Class II biological safety cabinet is recommended.

Biochemical procedures can be used to inactivate amplified products.

P2.1200 Other pre-amplification physical containment. Physical containment must include use of dedicated lab coats, gloves and disposable supplies. Frequent cleaning with dilute acid or bleach and/or UV treatment of work surfaces is recommended.

P2.1300 Equipment and Reagents

P2.1310 Equipment.

P2.1311 Use of dedicated equipment for pre-amplification procedures is recommended.

P2.1312 Use of dedicated pipettors is required. Positive displacement pipettes or filter-plugged tips are recommended.

P2.1313 Thermal cycling instruments must precisely and reproducibly maintain the appropriate temperature of samples. Accuracy of temperature control for samples should be verified on a regular basis.

P2.1320 Reagents.

P2.1321 All reagents (solutions containing one or multiple components) utilized in the amplification assay must be dispensed in aliquots for single use or reagents can be dispensed in aliquots for multiple use if documented to be free of contamination at each use. When reagents are combined to create a master mix, it is recommended that one critical component (e.g.Mg++) be left out of the aliquot.

P2.1322 Reagents (e.g., chemicals, enzymes) must be stored and utilized under conditions recommended by the manufacturer (i.e., storage temperature, test temperature, buffer, concentration). Reagents used for amplification must not be exposed to post-amplification work areas. The appropriate performance of each lot of reagent must be documented before results using these reagents are reported.

P2.1323 For commercial kits, the source, lot number, expiration date, and storage conditions must be documented. Reagents from different lots of kits must not be mixed. Each laboratory is responsible for the accuracy of typing. One possible approach for quality control is to test each reagent with a positive and negative control.

P2.1324 Primers must be stored under conditions that maintain specificity and sensitivity.

P2.1325 Methods that utilize two consecutive steps of logarithmic amplification are especially susceptible to errors related to PCR carryover (contamination) and special attention must be paid to containment of amplified products (e.g. physical separation, work flow and enhanced contamination monitoring). Standard 2.1100 applies to all components of the second amplification except template. Addition of the template for the second amplification must be physically separated from the pre-amplification work area and the post-amplification work area. Use of pipettors dedicated to each work area (i.e. first amplification, second amplification and analysis) is required.

P2.1400 Amplification templates

P2.1410 Specimens must be stored under conditions that do not result in artifacts or inhibition of the amplification reaction. Specimens must not be exposed to post-amplification work areas.

P2.1420 Nucleic acids should be prepared by a standard method that has been validated in the laboratory.

P2.1430 DNA or cDNA (from RNA templates) is satisfactory. DNA from any nucleated cells or RNA from any cells expressing the HLA product may be used. If RNA is used, appropriate positive controls for reverse transcription must be included.

P2.1440 Nucleic acids must be prepared and stored in a manner which does not result in artifacts or inhibition of the amplification reaction. The acceptable range for the amount of target must be specified and validated.

P2.1500 Primers

P2.1510 The specificity and sequence of primers must be defined. The HLA locus and allele(s) must be defined.

P2.1520 Conditions which influence the specificity or quantity of amplified product must be demonstrated to be satisfactory for each set of primers.

P2.1530 Reference material should be used to test and periodically reconfirm the specificity and product quantity of each lot of primers.

P2.1600 Contamination

P2.1610 Nucleic acid contamination must be monitored. Controls must be tested using the method that is routinely used to detect HLA types.

P2.1611 Negative controls (no nucleic acid) must be included in each amplification assay. Another negative control might include open tubes in the work area.

P2.1612 In order to minimize the detection of minor contaminants and the occurrence of stochastic fluctuation the number of cycles should be set at a level sufficient to detect the target nucleic acid but insufficient to detect small amounts (e.g., <10 molecules) of contaminating template.

P2.1613 Routine wipe tests of pre-amplification work areas must be performed. If amplified product is detected, the area must be cleaned to eliminate the contamination and measures must be taken to prevent future contamination.

P2.1700 Controls

P2.1710 The quantity of specific amplification products must be monitored (e.g., gel electrophoresis, hybridization).

P2.1720 Criteria for accepting or rejecting an amplification assay must be specified.

P2.1730 If presence of an amplified product is used as the end result, controls must be included to detect amplification failure in every amplification mixture. Amplification specificity must be monitored on a periodic basis.

P2.2000 Amplified Product (Nucleic Acid Targets)

P2.2100 Variation in the amount of amplified product must be monitored (e.g., hybidization with a consensus probe, gel electrophoresis). The acceptable range for the amount of available target must be specified.

P2.3000 Oligonucleotide Probes

P2.3100 HLA locus and allele(s) must be defined for each probe and template combination. Positive or negative probe hybridization must be defined for each probe with all possible combinations of alleles that are recognized by the W.H.O. provided that nucleotide sequences are readily available.

P2.3200 Probes must be stored under conditions which maintain specificity and sensitivity.

P2.3300 Probes must be utilized under empirically determined conditions that achieve the defined specificity. The specificity should be demonstrated and maintained for each lot of probe. Each lot of probes should be tested for specificity and product quantity using reference material under optimized conditions and reconfirmed periodically.

P2.3400 Hybridization must be carried out under empirically determined conditions that achieve the defined specificity.

P2.3500 The specificity of hybridization should be confirmed using positive and negative controls for hybridization with each probe. The controls should be capable of detecting cross-hybridization with closely related sequences.

P2.3600 Reuse of nucleic acids (probes or targets) bound to solid supports should only be undertaken after demonstrating that previous signals are no longer detectable.

P2.3700 Reuse of nucleic acids in solution (probes or targets) should only be undertaken with controls to ensure that the sensitivity and specificity of the assay are unaltered.

P2.3800 Incubators and water baths must be monitored for precise and accurate temperature maintenance every time the assay is performed.

P2.4000 Labeling of nucleic acids and detection

P2.4100 The specificity and sensitivity of the labeling and detection method must be established and reproducible.

P2.4200 The specificity and sensitivity must be maintained for each lot of reagents (e.g., antibodies, probes, indicator molecules).

P2.4300 Enzymes must be stored and utilized under conditions recommended by the manufacturer (i.e., storage temperature, test temperature, buffer, concentration) to ensure correct enzymatic activity. The enzymatic activity of each lot should be confirmed before use.

P2.5000 Analysis

P2.5100 Acceptable limits of signal intensity must be specified for positive and negative results. If these are not achieved, corrective action is required.

P2.5200 The method of assignment of types must be designated.

P2.5300 Two independent interpretations of primary data are recommended.

P2.5400 Reports must designate the type of assay (e.g., PCR/oligonucleotide), indicate the HLA locus, and define each type using W.H.O. nomenclature for alleles.

P2.5500 A permanent record of primary data must be retained for 2 years.
P2.6000 Nucleotide Sequencing

P2.6100 Sequencing Templates

Standards in P2.1400 must be followed for preparation of templates.

P2.6110 Templates must have sufficient specificity (e.g. locus or allele-specificity), quantity and quality to provide interpretable primary sequencing data. The method for preparing templates must reliably generate appropriate length sequencing templates that are free of inhibitors of subsequent reactions (e.g. primer extension) and free of contaminants that cause sequencing artifacts. Methods must ensure that preparation of templates does not alter the accuracy of the final sequence (e.g. mutations created during cloning, preferential amplification).

P2.6120 Reagents used in preparation of templates (e.g. enzymes, biochemicals) must be stored and utilized under conditions recommended by the manufacturer. The appropriate performance of each lot must be documented before results of tests using these reagents are reported.

P2.6200 Methods Utilizing Primer Extension

P2.6210 The specificity and general knowledge of the target sequence must be defined. The HLA locus and allele(s) must be defined.

P2.6220 Primers must be used under empirically determined conditions that achieve the defined specificity of amplification. The amplification conditions must be demonstrated by the laboratory to achieve defined specificity and must yield adequate quantity of specific product. Each lot of primer should be tested for specificity and product quantity using reference material (e.g.DNA) under routine conditions and reconfirmed periodically.

P2.6230 Conditions for primer extension (e.g. polymerase type, polymerase concentration, primer concentration, concentration of nucleoside triphosphates, concentration of terminators) must be appropriate for the template (e.g. length of sequence, GC content).

P2.6240 The specificity and sensitivity of the labeling and detection methods must be documented (e.g. demonstrating correct signal strength for a control sequence) in the laboratory before results are reported.

P2.6250 Satisfactory performance of each lot of reagent (e.g. nucleotides, enzymes) must be documented before results using these reagents are reported. Reagents must be stored under conditions that maintain optimal performance.

P2.6300 Electrophoresis

P2.6310 A sequencing standard must be run on every gel. The laboratory must establish scientifically and technically sound criteria for accepting each gel and each lane of a gel.

P2.6320 A permanent record of each electrophoretic run (e.g. electronic file, hard copy) must be retained for at least two years.

P2.6330 Satisfactory performance of each lot of reagents that influence the quality and accuracy of sequencing data of the gel (e.g. acrylamide, buffer and salt concentration) should be documented before results using these reagents are reported. Acceptable electrophoretic conditions (e.g. temperature, voltage, duration) must be established. Conditions should be recorded for each run. Reagents must be stored under conditions that maintain acceptable performance.

P2.6400 Nucleotide assignments

P2.6410 Criteria for acceptance of primary data must be established (e.g. correct assignments for nonpolymorphic positions, certain region of sequence, criteria for peak intensity, baseline fluctuation, signal-to-noise ratio and peak shapes). Validation might include sequencing of representatives of all polymorphic motifs that are frequently encountered in the routine sample population to detect sequence-specific artifacts. Sequencing of both strands of at least one representative of each polymorphic motif is recommended during validation. Established sequence-specific characteristics should be documented and utilized in routine interpretation of data.

P2.6420 Routine sequence assignments should be based on analysis of sequence data from complementary strands of DNA unless it is documented that the sequencing method consistently yields accurate sequence assignments using data from only one strand of DNA. If assignments are routinely based upon data from one strand of DNA, periodic confirmation of complementary strands is recommended. If base assignments are frequently difficult to interpret, routine sequencing of both strands is recommended. If a sequence suggests a novel allele or a rare combination of alleles, the sequences of both strands must be determined.

P2.6430 A scientifically sound and technically sound method must be established for interpretation, acceptance, and/or rejection of sequences from regions which are difficult to resolve (e.g. compression, ends).

P2.6440 Two independent interpretations of the primary data are recommended.

P2.6450 Automated systems and computer programs for nucleotide assignments must be validated prior to use.

P2.6500 Allele Assignments

P2.6510 HLA locus and alleles must be defined for each template/primer combination. Each unknown sequence must be compared with the sequences of all alleles that are recognized by the W.H.O. provided that the nucleotide sequences are readily available (i.e. in a locus-specific alignment in conjunction with the W.H.O. Nomenclature Committee for Factors of the HLA System which appears periodically in the public domain such as Tissue Antigens, the ASHI Web Pages or Human Immunology. Databases of sequences must be accurate and conform to the most recent compilation of sequences published in conjunction with the W.H.O.

P2.6520 Ambiguous combinations of alleles should be defined for each template/primer combination..

P2.6530 Methods must ensure that sequences contributed by amplification primers are not considered in the assignment of alleles.

P2.6540 Two independent assignments of alleles are recommended.

P2.6550 Automated systems and computer programs for allele assignments must be validated prior to use.

P2.6560 Reports must designate the type of assay, HLA locus, and define each type using W.H.O. Nomenclature for alleles. The laboratory must maintain records that define the sequence database utilized to interpret the primary data. This database must be updated periodically. If a determined sequence is ambiguous (i.e. more than one possible interpretation of available data) the report must indicate all possible allelic combinations.

P2.7000 Restriction fragment length polymorphism of amplified products

P2.7100 Restriction endonucleases

P2.7110 HLA locus and allele(s) must be defined for each RFLP type.

P2.7120 Enzymes must be stored and utilized under conditions recommended by the manufacturer (i.e., storage temperature, test temperature, buffer, concentration) to ensure correct enzymatic activity. The appropriate performance of each lot of enzyme must be documented before results using these reagents are reported.

P2.7130 When amplified DNA is digested, controls of amplified DNA which will produce fragments of known sizes must also be digested in parallel to monitor complete digestion.

P2.7200 Electrophoresis

P2.7210 Size markers of known sequence that produce discrete electrophoretic bands spanning and flanking the entire range of expected fragment sizes must be included in every run.

P2.7220 The amount of DNA/lane must not alter the rate of migration with respect to the migration of controls.

P2.7230 A permanent record (e.g., photograph, image) of each electrophoretic run must be retained as defined in C5.1000.

P2.7240 Amplified DNA should be incubated without restriction enzyme and analyzed by gel electropheresis to monitor marker integrity.

P2.7300 Analysis

P2.7310 Acceptable limits of signal intensity must be specified for positive and negative results. If these are not achieved, corrective action is required.

P2.7320 Appropriate migration patterns of control DNA and size markers are required.

P2.7330 The method of assignment of HLA types must be designated.

P2.7340 Two independent interpretations of primary data are recommended.

P2.7350 Reports must designate the type of assay (e.g., PCR/RFLP), indicate the HLA locus, and define each HLA type using W.H.O. nomenclature for alleles.

P2.8000 Typing using sequence-specific amplification

P2.8100 HLA locus and allele(s) must be defined for each primer combination. Positive or negative amplification must be defined for each primer mixture with all possible combinations of alleles that are recognized by the W.H.O. provided that nucleotide sequences are readily available.

P2.8200 Each amplification reaction must include procedures to detect technical failures (e.g. an internal control such as additional primers or templates that produce a product that can be distinguished from the typing product).

P2.8300 In each amplification assay (i.e. set up of amplification mixtures for one or more samples) controls should be used to detect contamination with previously amplified products (e.g., a special primer pair internal to all amplification products or a combination of primers to detect any DNA that could confound the typing result).

P2.8400 Primers must be utilized under empirically determined conditions that achieve the defined specificity for templates used in routine testing. Each set of primers must be tested for amplification specificity and product quantity using reference cells under optimized conditions. The frequency of testing each primer set must ensure that all primer pairs have appropriate sensitivity and specificity of amplification. The specificity and sensitivity must be maintained in heterozygous samples.

P2.8500 The specificity and sensitivity of the detection method must be established and reproducible.

P2.8600 Analysis

P2.8610 Acceptable qualitative limits of signal intensity must be specified for positive and negative results. If these are not achieved, corrective action is required.

P2.8620 The method of assignment of types must be designated.

P2.8630 Two independent interpretations of primary data are recommended.

P2.8640 Reports must designate the type of assay (e.g., SSP), indicate the HLA locus, and define each type using W.H.O. nomenclature for alleles.

P2.8650 A permanent record of primary data must be retained for 2 years.

P2.9000 Other Methods

P2.9100 If alternate methods (e.g.., SSCP, heteroduplex, DGGE) are used for HLA typing, established procedures must be defined and must include sufficient controls to ensure accurate assignment of types for every sample. All relevant standards from the above sections should be applied.

P2.9200 Automated systems and computer programs must be validated prior to use and tested routinely for accuracy and reproducibility of manipulations.