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What Keeps us up at Night The New Frontier in Blood Banking HEA Molecular Testing June 6, 2013 Sylvia M. Garza MT-P(AAB)

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Presentation on theme: "What Keeps us up at Night The New Frontier in Blood Banking HEA Molecular Testing June 6, 2013 Sylvia M. Garza MT-P(AAB)"— Presentation transcript:

1 What Keeps us up at Night The New Frontier in Blood Banking HEA Molecular Testing June 6, 2013 Sylvia M. Garza MT-P(AAB)

2 Objectives: 2 1.Define terms. 2.Discuss HEA molecular testing process. 3.Present case study demonstrating serological results that surprisingly differs from molecular results. 4. Review conclusions.

3 Terminology Definitions 101 Genotype- An individual’s actual genetic makeup. 2(p621) Genotype of a person is the complement of genes inherited by each person from his or her parents; the term is frequently also used to refer to the set of alleles at a single gene locus. 1(p334) Phenotype- The outward expression of genes (e.g., a blood type). On blood cells, serologically demonstrable antigens constitute the phenotype, except those sugar sites that are determined by transferases. 2(p629) Polymorphism for blood groups- the occurrence in the same population, of two or more alleles at one locus, each with appreciable (greater than 1%) frequency. 1(p335) Allele- One or two or more different genes that may occupy a specific locus on a chromosome. 2(613) 3

4 Terminology Definitions 101 cont. 4 Locus- The site of a gene on a chromosome. 2(p625) Chromosome- The structures within a nucleus that contain a linear thread of DNA, which transmits genetic information. Genes are arranged along the strand of DNA and constitute portions of the DNA. 2(p616) DNA (Deoxyribonucleic Acid)- The chemical basis of heredity and carrier of genetic information for all organisms, except RNA viruses. Structured as a double helix of polymers of nucleotides, each containing bases (A, T, C, and G), sugar deoxyribose, and three phosphates. They become monophosphates once they are built into DNA molecules. 2(p618)

5 BioArray Platform 5 The HEA BeadChip TM (Bioarray Solutions, Immucor) analyzes 24 polymorphisms associated with 38 Human Erythrocyte Antigens and phenotypic variants are included in the BioArray Solutions HEA BeadChip TM kit. 4(p4)

6 Principles of the Assay 6 The BioArray Solutions HEA BeadChip TM Kit uses the proprietary Elongation- mediated Multiplexed Analysis of Polymorphisms (eMAP®) technology to identify the presence or absence of the selected alleles associated with a phenotype. After multiplex PCR amplification and post-PCR, the single-stranded DNAs are incubated on the BeadChip TM array. Elongation reaction extends and incorporates fluorescently-labeled dNTP molecules only on those probes where the 3’ end matches the annealed DNA. Elongation products of alleles A and B are simultaneously detected by imaging the entire array.

7 Principles of the Assay cont. 7 In this method, each probe is covalently attached to a distinguishable bead type. A library of individual bead types contain all of the probes of interest. The library is immobilized in the BeadChip TM array, allowing for the detection of the polymorphisms of interest. 4

8 Principles of the Assay cont. 8 The BioArray Solution Array System (AIS400) is used to capture the fluorescent signal from individual beads in an image of the entire array, determine the identity of the bead by color and report the average signal intensity, coefficient of variance of the intensities, and the number of beads measured for each type of probe.

9 Principles of the Assay cont. 9 The HEA Analysis software in BioArray Solutions Information System (BASIS TM ) imports the raw intensity output, assesses the validity of the internal controls, and generates assay results.

10 Process 6 DNA Extraction Whole blood Multiplex PCR Amplification with Phosphorylated Primers Post PCR Processing Cleanup and Digestion Post PCR Processing Annealing and Elongation Assay Image Acquisition and BeadChip TM Analysis

11 Serology vs. Molecular Case Study 7 We followed an African American Woman (type A Neg) with Sickle Cell Anemia from and reviewed her history. Between , 41 PRBC’s were transfused with no reported transfusion reactions. 1.Patient Background To obtain the patient’s phenotype, we elected to perform the hypotonic saline wash technique due to the patient’s recent transfusions. Patient’s Serological phenotype: C+E-c+e+;M-N+S-s+;K- Fy(a-b-); Jk(a+b-) 2.Initial Serological Results

12 Serology vs. Molecular Case Study 8 2.Initial Serological Results cont. Summation of Results : -Anti-Fy a, Anti-Js a and Warm Autoantibody (autoanti-e) Anti-C, when the patient’s serotypes as C+ ???? 2012 The transfusing facility reports that another facility identified: Anti-E, Anti-K and Anti-C. So now the patient has Anti- Fy a, -Js a, -E, -K and Anti-C.

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15 Serology vs. Molecular Case Study 15 We need to look at the patient’s Genotype to see if the patient is homozygous for r ’ S. The (0) * may indicate a possible r ’ S So what is r ’ S ? Why do we care? So what is r ’ S ?  Patient’s who are R 2 r ’ S or homozygous r ’ S will be hr B- and can produce anti-hr B. 3(p65) In our case, this is why we care.  r ’ S phenotype also expresses partial C and these patients are at risk to form anti-C. 3(p65) Could this explain our anti-C?

16 LOOK!! L245V and G336C are homozygous for B

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18 Interpretation of HEA Genotyping by DNA Analysis 18 PolymorphismResultInterpretation RhCE L245VBBhr -B- also expresses partial C Very surprising. RhCE G336CBB FYA/FYBBB Hmmm, OK. GATABB [Red cells from patients that are FY*B but carry the GATA polymorphism are phenotypically Fy b neg. Because carriers of this single nucleotide polymorphism do express Fy b on cell types other than erythrocytes, they are not at risk for anti-Fy b production.] 3(p65)

19 Is Our Patient Remarkable? 19 Yes, this patient is remarkable. The 41 PRBC’s the patient was transfused didn’t cause any transfusion reactions. So we can deduce: The patient didn’t make anti-hr B, even thought the patient is hr B-. Or did make anti-hr B and the anti-hr B is clinically insignificant.

20 What Does This Mean Our Patient? 20 Our patient is rare in phenotype, and genotype but having made anti- E, -C, -Fya, and Jsa, locating antigen negative units should not be difficult. Patient’s Fyb is silenced This would eliminate the need for transfusing phenotypically Fy(a-b-) donor units. If in the future we had to recommend phenotypically negative units, we can now recommend genotypically negative units Fy(a-b+).

21 Conclusions 21  Genotypes differ from phenotypes.  Molecular testing helps supplement the limitations of serological techniques by using DNA to analyze and predict red cell phenotypes by providing genotype information.  Genotyping is helpful for providing information for problematic serological cases with complex or incomplete/questionable results.  Though this relatively new tool in the field of blood banking has not replaced serological testing, it continues to revolutionize and expand our knowledge of immunohematology.

22 References 22 1.Roback JD, Grossman BJ, Harris T, Hillyer CD. Technical Manual. 17 th ed. Bethesda, Maryland: American Association of Blood Banks (AABB); Harmening DM. Modern Blood Banking & Transfusion Practices. 6 th ed. Philadelphia, PA: F A Davis Company; Ness PM, Moulds JM. BeadChip Molecular Immunohematology, Toward Routine Donor and Patient Antigen Profiling by DNA Analysis. Springer Science+Business Media; HEA BeadChip TM Package Insert. Warren, NJ: BioArray Solutions; 2012.

23 Thank You for Your Time 23 Questions


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