Technical Bulletin No. 173

Transcription

1 CPAL Central Pennsylvania Alliance Laboratory Technical Bulletin No. 173 RBC Antigen Genotyping - New Test - February 11, 2019 Contact: Heather Habig, MLS (ASCP) CM, MB CM, Operations Manager, Molecular Diagnostics/Oncology Services, CPAL J Matthew Groeller, MPA(HCM), MT(ASCP), Operations Manager, Clinical Pathology, CPAL Jennifer Thebo, PhD, MT(ASCP), Director of Technical Operations and Scientific Affairs, CPAL Ordering Information and Suggested Codes: CPAL Test Name Lab Nexus Panel Code CPT Code HEA Blood Genotyping HEA RhCE Blood Genotyping RhCE RhD Blood Genotyping RhD Effective Date: Testing offered as of February 1, Performed: One day per week on dayshift. Samples will be extracted and held as they are received and tested on Thursdays. As volumes increase, the testing schedule will be adjusted accordingly. Testing Method: DNA Extraction; Elongation-mediated Multiplexed Analysis of Polymorphisms (emap ). Specimen Requirements: Whole blood collected in tubes containing EDTA as the anticoagulant. Minimum volume accepted: 1 ml. Whole blood is stable at 2 C to 8 C for up to one month. Fresher samples are preferable due to better DNA yield. Background: Human erythrocyte blood group antigens (HEA) are located on the membrane of the red blood cell. These surface markers are polymorphic protein and/or carbohydrate structures that are attached to lipid or protein. The PreciseType HEA BeadChip Kit contains twenty-four polymorphisms associated with thirty-five Human Erythrocyte Antigens and one with hemoglobinopathies. The PreciseType HEA Molecular BeadChip Test uses the proprietary Elongation-mediated Multiplexed Analysis of Polymorphisms (emap ) technology to identify the presence or absence of the selected alleles associated with a given phenotype. The BioArray Solutions RHD BeadChip Kit and RHCE BeadChip Kit also uses the Elongation-mediated Multiplexed Analysis of Polymorphisms (emap ) technology to identify the presence or absence of the selected alleles associated with a given phenotype. DNA extraction of whole blood and multiplex PCR analysis are used to isolate and amplify DNA specimen. Post-PCR processing is then performed using Clean-up Reagent and Lambda Exonuclease to produce singlestrand target DNAs or amplicons. The single-stranded DNAs or amplicons are incubated on the BeadChip array, allowing the annealing with the corresponding blood-group-specific probes. The BioArray Array

2 Imaging System is used to capture the fluorescent signal and processes the data generated. The BioArray Solutions Information System (BASIS ) analyzes the data and generates assay results. Contents of Panels: For a list of genetic markers contained in each panel and the expected phenotypes, please see the tables in the Appendix of this document. Validation: Included in the validation were method correlations of the HEA, RhD, and RhCE assays using vendor-provided DNA panels and whole blood samples obtained from members and reference laboratories used by members. The results were within acceptable parameters and are available onsite for review upon request. Limitations: 1. Any results that are generated must be interpreted in conjunction with other clinical or laboratory findings. 2. BioArray Solutions RHD and RHCE BeadChip kits are for Research Use Only. These tests are not intended for clinical diagnosis or as the sole means for patient management decisions. 3. False positive and/or invalid HEA results may be generated in rare cases where a sample contains examples of molecular events that affect the blood-group antigen expression and phenotypes and the nucleotide changes associated with these events are not explicitly monitored by the assay. Examples include DNAsequence variations including premature stop codon, SNP leading to missense change in amino acid, hybrid genes, modifying genes; changes at the RNA transcription level including alternative splicing; reduced protein expression, etc. Known phenotypes are Knull, JKnull (JKnull has a prevalence of up to 9% among Polynesians), Rhnull, Rh hybrids, Kmod, Co(a-,b-), In(Lu), Lu(a-,b-), and GP hybrids. Presence of a c.179_180del (Ser60fs) mutation linked with the Fy(b) allele may change the Fy(b) antigen expression and lead to a false positive result. 4. False negative and/or HEA invalid results may be generated when unanticipated rare mutation(s) affecting the primer or probe binding cause allele and/or amplicon dropout. 5. The RHD kit is unable to differentiate between homozygous and hemizygous forms of alleles. 6. The RHD kit is unable to detect hybrid alleles in a heterozygous combination with other alleles, when the hybrid allele does not have any characteristic markers outside the hybrid region. 7. The RHCE assay is unable to detect RHCE-RHD hybrids, except for CeRN. The CeRN allele can only be detected in the homozygous state. 8. Certain alleles cannot be detected using the RHCE assay because of the absence of relevant markers in the RHCE BeadChip panel. These alleles may be mistyped as alleles that are covered by the RHCE assay. References: 1. Immucor BioArray PreciseType HEA Molecular BeadChip Test doc EN Rev. C Oct Immucor BioArray PreciseType RHCE Molecular BeadChip Test doc EN Rev. G Oct Immucor BioArray PreciseType RHD Molecular BeadChip Test doc EN Rev. E Oct Immucor ETC AIS Exposure Test P/N Rev. A Date of Revision: July Immucor PreciseType HEA Test User Training Guide, doc Rev B, Issued on: February 11, 2019 Page 2 of 6

3 APPENDIX Genetic Markers for Red Blood Cell Antigens in the PreciseType HEA Test: Issued on: February 11, 2019 Page 3 of 6

4 Genetic markers in the RHD BeadChip Kit: Issued on: February 11, 2019 Page 4 of 6

5 Phenotypical variants detected by the RHD BeadChip kit: Table 3c: D Negative Hybrid Alleles: Hybrids with RHD negative phenotypes RHCE(1-3)-D(4-10) RHD-CE(3-7)-D RHD-CE(3-9)-D RHD-CE(4-7)-D DIIIa-CE(4-7)-D Issued on: February 11, 2019 Page 5 of 6

6 Genetic markers in the RHCE BeadChip Kit: Phenotypical variants detected by the RHCE BeadChip kit: Notes: The presence of alleles marked with may suggest the presence of (C)ces or r s haplotype. For confirmation, test the sample for presence of DIIIa-CE(4-7)-D using the RHD BeadChip kit. The above tables are not all inclusive of all possible RHCE genetic variations. See the Limitations section for RHCE variations not covered by this test. Issued on: February 11, 2019 Page 6 of 6