BLOOD GROUP SUBSTANCES
1 VALIDATION OF GENOTYPING PROTOCOLS FOR DIAGNOSTIC USE 2 HIGH-RESOLUTION MELTING ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISMS 3 HIGH-SPEED DROPLET-ALLELE-SPECIFIC POLYMERASE CHAIN REACTION FOR GENOTYPING OF SINGLE NUCLEOTIDE POLYMORPHISMS 4 BLOOD GROUPING BASED ON PCR METHODS AND AGAROSE GEL ELECTROPHORESIS 5 PARALLEL DONOR GENOTYPING FOR 46 SELECTED BLOOD GROUP AND 4 HUMAN PLATELET ANTIGENS USING HIGH-THROUGHPUT MALDI-TOF MASS SPECTROMETRY 6 PCR WITH SEQUENCE-SPECIFIC PRIMERS FOR TYPING OF DIALLELIC BLOOD GROUPS 7 HIGH-RESOLUTION MELTING ANALYSIS FOR GENOTYPING DUFFY BLOOD GROUP ANTIGENS 8 MOLECULAR RHD-RHCE ANALYSIS BY MULTIPLEX PCR OF SHORT FLUORESCENT FRAGMENTS 9 MICROARRAYS IN BLOOD GROUP GENOTYPING 10 NEXT-GENERATION SEQUENCING FOR ANTENATAL PREDICTION OF KEL1 BLOOD GROUP STATUS 11 454-SEQUENCING™ FOR THE KEL, JR, AND LAN BLOOD GROUPS 12 NONINVASIVE PRENATAL BLOOD GROUP GENOTYPING 13 GENOTYPING OF HUMAN PLATELET ANTIGENS BY BEADCHIP MICROARRAY TECHNOLOGY CONTENTS 14 SEQUENCE-BASED TYPING FOR PLATELET ALLOANTIGENS 15 MINIATURIZED TECHNOLOGY FOR DNA TYPING: CASSETTE PCR 16 GENO- AND PHENOTYPING OF HUMAN NEUTROPHIL ANTIGENS 17 ALLELIC DISCRIMINATION BY TAQMAN-PCR FOR GENOTYPING OF HUMAN NEUTROPHIL ANTIGENS 18 NOVEL APPROACHES AND TECHNOLOGIES IN MOLECULAR HLA TYPING 19 LUMINEX-BASED METHODS IN HIGH-RESOLUTION HLA TYPING 20 IN SILICO HLA TYPING USING STANDARD RNA-SEQ SEQUENCE READS CHAPTER VALIDATION OF GENOTYPING PROTOCOLS FOR DIAGNOSTIC USE ABSTRACT THIS CHAPTER CONTAINS SOME ADVICE FOR ? RST VALIDATING A NEW GENOTYPING METHOD AND THEN CONTINUING TO ENSURE THAT IT PERFORMS WELL WHEN ADOPTED FOR ROUTINE USE IT SHOULD BE APPLICABLE TO MOST OF THE GENOTYP- ING METHODS DESCRIBED IN THIS VOLUME; HOWEVER, BECAUSE THE RANGE OF TECHNIQUES IS SO WIDE AND THESE RECOMMENDATIONS HAVE BEEN WRITTEN WITH PCR AMPLI? CATION IN MIND, THE READER MAY NEED TO ADAPT SOME OF THE RECOMMENDATIONS FOR NON-PCR BASED TESTS IT DOES NOT INCLUDE ANY ADVICE ON HOW TO HANDLE UNSCREENED HUMAN SAMPLES WITHIN THE LABORATORY, AND IT ASSUMES THAT THE LABORATORY ALREADY HAS ESTABLISHED GOOD PRACTICE FOR DNA AMPLI? CATION AND THE AVOIDANCE OF CONTAMINATION KEY WORDS GENOTYPING , PROTOCOL , VALIDATION 1 INITIAL VALIDATION OF A NEW GENOTYPING METHOD 1 IT IS ESSENTIAL TO START WITH A SMALL PANEL (4–6) OF DNA SAMPLES WHICH HAVE BEEN PREPARED RECENTLY BY A ROUTINE METHOD THEY SHOULD BE AVAILABLE IN BULK SO THAT THE SAME SAMPLES CAN BE USED REPEATEDLY FOR THE INITIAL RUNS; THUS ANY DIFFERENCES OBSERVED FROM RUN TO RUN CAN BE ASSUMED TO BE RELATED TO VARYING CONDITIONS RATHER THAN VARIATIONS IN SAMPLE QUALITY OR DIFFERING GENOTYPES 2 SAMPLES FROM THE SAME DONORS SHOULD HAVE BEEN PHENOTYPED AS FAR AS POSSIBLE AND GENOTYPED BY A TECHNIQUE ALREADY ESTAB- LISHED IN THE SAME LABORATORY OR BY AN EXTERNAL LABORATORY IF THIS IS A NEW AREA FOR THE DEVELOPING LAB THE PANEL SHOULD INCLUDE AS WIDE A RANGE OF GENOTYPES AS POSSIBLE IN SUCH A SMALL PANEL 3 IF THE GENOTYPING TECHNIQUE INVOLVES OLIGONUCLEOTIDE PRIMERS OR PROBES, THEN THE SEQUENCES SHOULD BE CHECKED CAREFULLY TO SEE IF THEIR FUNCTION COULD BE AFFECTED BY UNDERLYING SNPS (OTHER THAN THE ONE WHICH MAY BE THE TARGET OF THE GENOTYPING TEST) ADDITIONAL SNPS THAT ARE SITUATED SEVERAL BASES FROM THE 3? END OF PRIMER SEQUENCES HAVE BEEN SHOWN TO HAVE SIGNI? CANT EFFECTS ON EF? CIENCY OF AMPLI? CATION 4 THE SMALL PANEL OF SAMPLES SHOULD BE USED IN ALL INITIAL TESTS CARRIED OUT TO GET THE TECHNIQUE WORKING FOR EXAMPLE, IF THE GENOTYPING ASSAY INVOLVES PCR, THEN THIS STAGE WILL INVOLVE MULTIPLE EXPERIMENTS WHERE CONCENTRATIONS OF PRIMERS AND TEMPLATE (AND POSSIBLY BUFFER AND DNTPS) ARE VARIED, USUALLY ONE AT A TIME PCR CONDITIONS WILL BE ESTABLISHED PRIMARILY BY VARYING ANNEALING TEMPERATURE(S) AND CYCLE NUMBER IF THE NEW GENOTYPING ASSAY IS REQUIRED TO RUN UNDER THE SAME CONDITIONS AS AN EXISTING TECHNIQUE (I E , “MULTIPLEXED”), THEN POSSIBLY BOTH SETS OF CONDITIONS WILL HAVE TO BE VARIED IN ORDER TO ACHIEVE A COMMON PROTOCOL 5 WHEN THE NEW TECHNIQUE APPEARS TO WORK AND THE EXPECTED RESULTS CAN BE RELIABLY OBTAINED FROM THE SMALL PANEL, THEN THE SAME SAMPLES CAN BE TESTED IN DILUTION TO DE? NE A WORKING RANGE FOR ACCEPTABLE DNA CONCENTRATION IN SUBSEQUENT PATIENT SAMPLES DEGRADED DNA IS NOT THE SAME PROBLEM AS LOW DNA CONCENTRATION AND ANOTHER MEASURE OF DNA QUALITY SHOULD BE INTEGRATED IF DEGRADATION IS SUSPECTED 6 FOR TECHNIQUES INVOLVING PCR, USERS COULD EXPLORE ALIQUOTING AND FREEZING OF PRIMER MIXES AT THIS STAGE IF THEY CAN BE STORED READY FOR USE IN INDIVIDUAL TUBES, EITHER IN STRIPS OR PLATES, THEN TIME WILL BE SAVED AND MISTAKES MADE WHILST SETTING UP THE TEST WILL BE LESS LIKELY 7 WHEN ALL PARAMETERS HAVE BEEN EXPLORED AND A ROBUST SET OF CONDITIONS HAVE BEEN ADOPTED, THEN A LARGE-SCALE VALIDATION STUDY SHOULD BE CARRIED OUT AND DOCUMENTED AT LEAST BLINDED SAMPLES, INCLUDING AT LEAST ? VE EXAMPLES OF EACH COM- MON GENOTYPE, SHOULD BE TESTED AND COMPARED WITH RESULTS PREVIOUSLY OBTAINED BY ANOTHER METHOD (INTERNAL OR EXTERNAL) THE PANEL SHOULD INCLUDE AS MANY HETEROZYGOTES AS POSSIBLE AS THESE ARE MORE PRONE TO ERROR THAN HOMOZYGOTES RARE GENO- TYPES SHOULD ALSO BE INCLUDED IN THE PANEL IF POSSIBLE THE RACIAL MIX IN THE INTENDED PATIENT POPULATION SHOULD BE CONSIDERED AND IF NECESSARY ADDITIONAL SAMPLES OF RARE GENOTYPES SHOULD BE OBTAINED FROM AN EXTERNAL LABORATORY 8 IF EITHER CE-MARKED OR WHO ENDORSED DNA REFERENCE MATE- RIALS ARE AVAILABLE, THEN THESE SHOULD ALSO BE TESTED AT THIS STAGE 2 ONGOING VALIDATION OF AN ESTABLISHED GENOTYPING METHOD CONTINUING VALIDATION OF A METHOD DEPENDS ON THE FREQUENCY OF TESTING AND THE SCALE OF THE CHANGE BEING MADE AND THE FOLLOWING IS ONLY A GUIDE THERE ARE NO SPECI? C RECOMMENDATIONS MADE FOR BLOOD GROUP SUBSTANCES THE NUMBER OF SAMPLES REQUIRED FOR STEPS 1 AND 2 BECAUSE THIS DEPENDS ON THE TOTAL NUMBER OF POSSIBLE GENOTYPES; FOR EXAMPLE, A SMALL PANEL WOULD BE REQUIRED TO CONTROL THE REACTIONS DETECTING ONLY ONE SNP BUT IF THE METHOD SIMULTANEOUSLY DETECTS MULTIPLE SNPS, THEN MORE CONTROLS WOULD BE REQUIRED 1 A SMALL SET OF CONTROLS, INCORPORATING A RANGE OF GENOTYPES WITH AS MANY HETEROZYGOTES AS POSSIBLE, SHOULD BE INCLUDED IN EACH ASSAY IF REASONABLY PRACTICABLE 2 A LARGER PANEL, INCORPORATING EACH GENOTYPE AT LEAST ONCE, SHOULD BE USED TO VALIDATE NEW BATCHES OF PRIMERS, NEW BULK PREPARATIONS OF PRIMER MIXES, OR NEW BATCHES OF TAQ, DNTPS, OR PCR REACTION BUFFER 3 IF A MAJOR CHANGE IS MADE, FOR EXAMPLE AN ALTERATION TO THE CYCLING PARAMETERS OR A NEW PCR MACHINE, THEN THE TECHNIQUE SHOULD BE REVALIDATED USING A PANEL SIMILAR TO THAT DESCRIBED IN SUBHEADING 1 , STEP 7 4 IT IS IMPORTANT TO RECORD THE BATCH NUMBERS OF ALL REAGENTS USED IN EACH GENOTYPING RUN AND TO KEEP A LOG OF THE DATES WHEN CRITICAL REAGENTS ARE CHANGED IT IS ALSO IMPORTANT TO CARRY OUT REGULAR TEMPERATURE CALIBRATION OF ANY THERMOCYCLER BLOCK AFTER CONSULTATION WITH THE MANUFACTURER CONCERNING THE REC- OMMENDED INTERVAL FOR SUCH TESTING 5 IF POSSIBLE AUTOMATED READING, INTERPRETATION, AND RECORDING OF RESULTS SHOULD BE INCORPORATED INTO THE METHOD RATHER THAN OPERATOR-DEPENDENT STEPS, IN ORDER TO LOWER THE OPERATOR ERROR RATE HOWEVER, IT IS IMPORTANT TO STORE DOCUMENTARY EVIDENCE OF THE ACTUAL TEST RESULT (E G , PHOTO OF GEL, SCREENSHOT OF TRACE) RATHER THAN JUST THE INTERPRETED GENOTYPE RESULT IN THIS WAY RESULTS CAN BE CHECKED AND SUBTLE CHANGES IN PERFORMANCE CAN BE DETECTED 6 THE LABORATORY SHOULD JOIN AN EXTERNAL QA SCHEME IF ONE IS AVAILABLE FOR THE GENOTYPES INVOLVED BLOOD GROUP SUBSTANCES