Is it time to re-evaluate the gold standard in red cell antigen typing?

Transcription

1 Is it time to re-evaluate the gold standard in red cell antigen typing? Susan T. Johnson, MSTM, MT(ASCP)SBB American Society of Clinical Laboratory Science Central New England May 9, GLOBCC 8MAY2015

2 Objectives List molecular events that result in red cell antigen expression. Describe application of molecular testing in immunohematology. Discuss advantages and limitations of red cell phenotyping and genotyping. 2 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

3 Strengths of Serology (S.O.S.) Benchmark / Known Entity Detects phenotype Antigen & Antibody Understand clinical relevance Real-time testing Automated Inexpensive 3 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

4 Antigen Typing ABO/Rh Type Rh phenotype Other blood group antigens Discrepant results using different reagents Variant antigens weak positive reactions Variant antigens unexpected positive reactions 4 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

5 Polyclonal Antibodies Normal immune response stimulates the production of many different immunoglobulins recognizing different epitopes on antigen Antigen JR Storry 5 = immunoglobulin Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

6 Monoclonal Antibody Monoclonal antibody is specific for 1 epitope Antigen JR Storry 6 = immunoglobulin Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

7 Monoclonal versus Polyclonal Ag Antigen expansion B cells Antibodies JR Storry 7 Monoclonal Polyclonal Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

8 Case 1 45 y/o African American male Blood donor 9 previous donations, typed as B, RBC unit is labeled as B Trainee finds this result Anti-A Anti-B A 1 cells B cells Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

9 Case 1 Investigation Repeat ABO type Anti-A Anti-B A 1 cells B cells Type with a different mo Anti-A Anti-A Anti-B A 1 cells B cells (mo 2) Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

10 B ANTIGEN B gene -3-D-galactosyltransferase Gal 10 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

11 A ANTIGEN Super B gene -3-N-acetylgalactosaminyltransferase Extra Antigen GalNAC 11 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

12 ABO Gene to Gene Product (Transferase) Exons 9q34. RNA mrna G. Denomme 5 UTR 3 UTR * * * * Globular Head GalNAc1 3Gal -R 2 Fuc Gal1 3Gal -R 2 Fuc 354 amino acids G. Denomme 12 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping A B

13 Case 1 Investigation Type with polyclonal Anti-A Anti-A Anti-B A 1 cells B cells Test with Group B plasma Donor 1 Donor 2 Donor Donor is a B! 13 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

14 Rh Typing Weak Expression of RhD C in trans with RHD Weak D Types Partial D D epitopes on RhCE protein Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

15 RH Genes Rh Positive Chromosome 1 Locus 1 Locus 2 RHD RHCE D C, E, c, e 15 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

16 Molecular Types Associated with & Impacting RhD Expression RHD/RHCE Hybrid Alleles Single nucleotide polymorphisms (exchanges) Multiple nucleotide exchanges Deletions or insertions Splice site mutations Premature stop codons 16 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

17 Anti-D Monoclonal Reagent Types Blend of monoclonal & polyclonal antibodies Blend of two or more monoclonal antibodies, each secreted by a different cell line IgG or IgM, or combination of IgG + IgM Why? D antigen has >30 different epitopes Variant D antigens 17 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

18 Case 2 27 y/o Caucasian female Prenatal evaluation Rh phenotype: C+E-c+e+ 18 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

19 Case 2 Anti-D Patient RBCs Reagent IgM IgG IS IAT GammaClone GAMA401 F8D Seraclone BS232 BS H41 11B7 Immucor-5 TH28 MS26 W+ 2+ Ortho Bioclone Ortho (ID-MTS) MAD2 Human polyclonal 0 3+ MS Are they RhD Positive or RhD Negative? 19 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

20 Other Typing Reagents Rh Anti-C, Anti-E, Anti-c, Anti-e Others Anti-Jk a, -Jk b, -Fy a, -Fy b, -K, -S, -s, -M All may show discrepant results! 20 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

21 Case 3 66 year-old African American GI bleed, 4 pregnancies; history of transfusion O, D+C-E+c+e+ Anti-e, -Jk b Currently no transfused RBCs present and autologous control is negative 21 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

22 Problem Patient s RBCs are e+ Patient has made apparent alloanti-e A familiar problem! 22 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

23 Test Patient s RBCs with Anti-e Anti-e reagent (clone/s) Gamma-Clone (MS16, MS21, MS63) Patient (E+e+) Control (E+e+) Control (E+e-) Immucor Series 1 (MS16) Ortho Bioclone (MS16) Polyclonal 1+/2+ 4+/4+ 0 Slide 23

24 Other Reagent Typing Problems Polyclonal (Immucor) Monoclonal (Ortho) Sample Anti-Jk a Anti-Jk b Anti-Jk a MS15 Anti-Jk b MS to + W to NT From: Whorley T, et al. Transfusion 2009; 49S: 48A Abstract (S E) 24 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

25 Reagent Grade Antisera Not Available/Not Effective Anti-Do a Anti-Do b Anti-Js a U variants Anti-hr B Anti-hr S Anti-V Anti-VS 25 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

26 DNA Technology Red Cell Genotyping 4 results (A, C, G, T) G Denomme 26 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

27 Blood Group Systems Review Systems 33 Gene Loci 37 Antigens 294 Alleles >2,000 Plus 42 antigens in Collections, Low Series, and High Series 27 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

28 Molecular Types Associated with Blood Group Antigen Expression Single nucleotide polymorphisms (exchanges) Gene rearrangement with recombination and conversion events Multiple nucleotide exchanges Deletions or insertions Splice site mutations Premature stop codons Duplication events Changes to Regulator/Modifying Genes 28 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

29 Single Nucleotide Polymorphisms Single nucleotide polymorphisms (changes) - SNPs - are most common genetic change leading to the expression of blood group antigens G G C A T T C G G G C A T T G G Amino Acid Change In DNA the Purine bases are Adenine and Guanine, while the Pyrimidines are Thymine and Cytosine Blood Group Antigen e.g. Jk a /Jk b 29 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

30 Molecular Techniques Red Cell Genotyping Allows for mass screening for antigen negative blood and rare units. Ability to genotype patients for common antigens C, c, E, e, M, N, S, s, K, k, Fy a, Fy b, Jk a, Jk b, Js a, Js b, Kp a, Kp b, Do a, Do b, Lu a, Lu b, Co a, Co b, Di a, Di b, Yt a, Yt b U, Uvar (Bold indicates SNPs) 30 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

31 Molecular Techniques RHCE Variants C, c, E, e, (including ce s ), hr S, hr B, altered C (r S ), V, VS, Crawford, C w, C x 31 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

32 Patient Red Cell Genotyping Can be performed regardless of transfusion history and/or if the patient has a positive DAT. Only a small EDTA sample is needed to perform DNA isolation and genotyping (require white cells). Obtain a large amount of information without the use of many serological reagents. Can detect variant and null alleles that can be further characterized. Although not routinely available on-site, can be performed and interpreted within only a few hours 32 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

33 Antibody Detection 33 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

34 Antibody Detection Test Tube Enhancement Methods EE Serum to Cell Ratio 4 to 1 Incubation time Albumin LISS PEG Enzymes Use RBCs with double-dose antigen 34 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

35 Fluid-Phase Methods Test Tube Methods Microplates Column Agglutination Technology Gel Beads 35 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

36 Serologic Behavior of Antibodies Test Tube Methods IS &/or RT 37C IAT MOST FREQUENT -Le a, -Le b, -P 1, -M, -N, -Lu a, Cold Autoantibodies (-I, -IH, -i) OCCASIONALLY REACT -K, -D, -C, -E, -c, -e MOST FREQUENT -D, -C, -E, -c, -e, -K, Others HEMOLYSIS -Le a, -Le b, -Jk a, -Jk b, -Vel, -PP 1 P k AGGLUTINATION AT 37C ONLY -K, -E 36 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping MOST FREQUENT -D, -C, -E, -c, -e, -K, -Jk a, -Jk b, -Fy a, -Fy b, -S,-s LESS FREQUENT Knops Antibodies, -Lu b, -Sd a, -Bgs Antibodies to high incidence antigens, Warm autoantibodies, Cold-reactive antibody binding C3

37 Antibodies don t read the books! Anti-Fy a, Anti-Jk a, Anti-Jk b positive at IS, 37C and IAT Anti-k reacts in saline, but not in PEG Many Other Examples! 37 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

38 Serologic Behavior of Antibodies CAT in USA Buffered Card RT Buffered Card 37C IgG Card IAT MOST FREQUENT *-Le a, -Le b, -P 1, *-M, -N, *-Lu a, *Cold Autoantibodies (-I, -IH, -i) *Mixed Cell OCCASIONALLY REACT -K, -D, -C, -E, -c, -e MOST FREQUENT -D, -C, -E, -c, -e, -K, Others, -M HEMOLYSIS -Le a, -Le b, -Jk a, -Jk b, -Vel, -PP 1 P k AGGLUTINATION AT 37C ONLY -K, -E?? MOST FREQUENT -D, -C, -E, -c, -e, -K, -Jk a, -Jk b, -Fy a, -Fy b, -S,-s LESS FREQUENT Knops Antibodies, -Lu b, -Sd a, -Bgs Antibodies to high incidence antigens, Warm autoantibodies, *-Le a, -Le b, *-M, Cold Autoantibodies (-I, -IH, -i) *Mixed Cell 38 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

39 Case 4 27 y/o Caucasian female History of unidentified antibody Planning future pregnancy 39 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

40 Antibody Identification Panel at Hospital Rh MNS P Lewis Kell Duffy Kidd Gel D C E c e f M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b IAT A 0 40 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

41 Antibody Identification Panel at IRL Rh MNS P Lewis Kell Duffy Kidd Saline D C E c e f M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b IS 30 37C IAT A DAT 0 41 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

42 Antibody Identification Panel at IRL Rh MNS P Lewis Kell Duffy Kidd PEG D C E c e f M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b 15 37C NH NH NH NH NH NH NH NH NH 0 A NH 0 IAT 42 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

43 Antibody Identification Panel at IRL Rh MNS P Lewis Kell Duffy Kidd Gel D C E c e f M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b 15 37C NH NH NH NH NH NH NH NH NH 1 A NH w+ IAT 43 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

44 Selected Cells D C E c e K k Fy a Fy b Jk a Jk b S s Gel Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

45 Phenotype Results RBCs Anti-K Anti-k #1 Anti-k #2 Anti-Kp a Patient Pos Ct Neg Ct Phenotype: K+k+Kp(a-) Anti-k reactive only by Gel IAT Auto or alloantibody? 45 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

46 Solid-Phase Methods 46 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

47 Solid Phase Red Cell Adherance Patient Antibody Indicator RBCs Negative Positive 47 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

48 Protein A Coated Wells Plasma or serum added to well, followed by MLB (modified LISS) and screening cells to protein A coated well Incubate 20 min at 37C Screening RBC s are coated with patient antibodies, if antibodies are present in serum/plasma 48 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

49 Protein A Coated Wells Wells are washed with PBS Anti-IgG is added to the wells Sensitized RBC s and Anti-IgG bind with Protein A on well surface Unsensitized RBC s form button at bottom of well 49 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

50 SOLID PHASE Unique Characteristics Enhanced detection of anti-d & anti-jk Sensitivity is high for all antibodies Warm autoantibodies detected Less variability in positive reactivity May not detect agglutinating antibodies 50 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

51 Serologic Behavior of Antibodies SP Buffered Card RT Buffered Card 37C IAT MOST FREQUENT -Le a, -Le b, -P 1, -M, -N, -Lu a, Cold Autoantibodies (-I, -IH, -i) OCCASIONALLY REACT -K, -D, -C, -E, -c, -e MOST FREQUENT -D, -C, -E, -c, -e, -K, Others, -M HEMOLYSIS -Le a, -Le b, -Jk a, -Jk b, -Vel, -PP 1 P k AGGLUTINATION AT 37C ONLY -K, -E?? MOST FREQUENT -D, -C, -E, -c, -e, -K, -Jk a, -Jk b, -Fy a, -Fy b, -S,-s LESS FREQUENT Knops Antibodies, -Lu b, -Sd a, -Bgs Antibodies to high incidence antigens, Warm autoantibodies, *-Le a, -Le b, *-M, Cold Autoantibodies (-I, -IH, -i) 51 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

52 Passive Anti-E in a Pediatric Patient Not Detected by Routine Donor Screening Using Anti-IgG Solidscreen II or Gel Test MG Marchan, LA Leszczynski, JS Curnes, JJ Rauter, RC Punzalan, ST Johnson, BloodCenter of Wisconsin, Inc., Milwaukee, WI SP344 Transfusion 2007, Vol 47 Supplement 52 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

53 Case History 6-month old female weighing 7.1 kg. After a negative pre-transfusion antibody detection test, she received 60 ml of leukocyte-reduced whole blood during cardiac bypass surgery. (Only previous transfusion was 1 leukocyte-reduced RBC 4 mos. prior to surgery). 24 hours after WB transfusion, a new request for transfusion required testing of a new sample. 53 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

54 Serological Testing on Patient & Donor using PeG Test Tube Method Patient Transfused Whole Blood ANTIBODY DETECTION TEST Negative Pretransfusion Posttransfusion Positive (1+) Positive (3+) Autocontrol N/A Negative Negative ANTIBODY N/A Anti-E Anti-E IDENTIFICATION PHENOTYPE N/A E Negative E Negative 54 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

55 Serological Testing on Donor Samples Test Method Previous 13 Donations Current Donation (Implicated Unit) Subsequent Donation ID-MTS GEL test Negative* Negative Negative TANGO Anti-IgG Solidscreen II N/A Negative Negative PeG IAT N/A Positive (3+) Positive (3+) Anti-E passively acquired from donor, only reactive in a PEG IAT 55 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

56 Immunohematology 2006;22: No Method Detects All Antibodies!! 56 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

57 Case 5 21 y/o Indian male Thalessemia Transfused since 3 mos. old History of anti-e detected in 1996 ATR 3 months ago Anti-c suspected + additional weak positive reactions 57 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

58 Dealing with Current Antibody Identification Problems With complex workups, antigen typing is performed to help aid in antibody identification. Patient antigen typing helps confirm the presence of an antibody, by proving the patient is antigen negative for the antibody in their serum. If a patient has been transfused within 3 months of testing or has a positive DAT, antigen typing cannot be performed using many routine serological methods. RCG can be performed regardless of transfusion history and a positive DAT. 58 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

59 Aiding in Future Antibody Identification Problems By knowing the patient s genotype, the next time the patient presents as a problem, the transfusion service or IRL will know what antibodies the patient can make. Therefore enhancing their problem solving capabilities. Most useful if patient has multiple occurrences of making antibodies. Caution!! Antigen negative doesn t automatically mean patient has made antibody. 59 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

60 Case 5 21 y/o Indian male Thalessemia Transfused since 3 mos. old History of anti-e detected in 1996 ATR 3 months ago Anti-c suspected + additional weak positive reactions Genotype C+E-c-e+; K-k+;Fy(a+b-);Jk(a+b+) 60 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

61 Preventing Alloimmunization Genotype matching a patient and donor decreases chance of alloimmunization dramatically. Patient s at risk for alloimmunization - Previously alloimmunized patients - Transfusion-dependent diseases (Chronically transfused patients) Sickle Cell patients Thalassemia Myelodysplastic Syndrome (MDS) Warm Autoimmune Hemolytic Anemia (WAIHA) 61 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

62 Preventing Alloimmunization Multiply Transfused Patients Patients at Risk of Producing Alloantibodies Patients who have already produced alloantibodies or autoantibody Use data for current & future antibody identification work-ups Obtain a Complete Red Cell Genotype Provide complete genotype matched blood Save time/resources for future antibody identification work-ups Outcome The future of transfusion medicine? prevent alloimmunization 62 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

63 Limitations of Genotyping Turnaround Time needs to be faster Data from genotyping needs to be incorporated into donor and hospital systems Must be able to query inventory Must have a system for resolving discrepant results 63 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

64 Success of Serology Serology tells us Antigen is present or not present on RBCs Variants may be present when discrepant results are found To Listen to results and investigate Antibodies are present in plasma/serum 64 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

65 SOS 65 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

66 SOS Limitations of Serology Weak antigen difficult to detect Reagent grade antisera not available for all specificities Miss some antibodies Alloantibody vs. Autoantibody? Antigen positive with corresponding antibody Does not predict significance of antibodies Does not prevent alloimmunization 66 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping

67 Please let go. It s okay, really!!

68 Thank You! 68 Is it time to re-evaluate the gold standard in red cell antigen typing? S Johnson May 9, 2013 Red Cell Genotyping