Why We Do What We Do: Serologic Testing 10/20/2007 2:00PM 5:30PM

Transcription

1 Why We Do What We Do: Serologic Testing 1/2/27 2:PM 5:3PM

2 Event Outline Event Title: 53-TC Why We Do What We Do: Serologic Testing Event Directors: Lieta Maffei Lieta Maffei Event Date: Saturday, October 2, 27, 2: PM to 5:3 PM Presenters: W. John Judd Susan Johnson Theresa Downs Debra Bailey Time Speaker Presentation 2: PM to 2:3 PM Susan Johnson 3:3 PM to 4: PM Debra Bailey 4: PM to 4:3 PM Theresa Downs Why Are We Performing the ABO Test and What Are We Detecting Why Do We Perform an Antibody Detection Test and How it Can Help with the Antibody Identification Which Crossmatch Do I Do Now? 6629 W. John Judd What to do about Weak D?

3 EVENT FACULTY LIST Event Title: 53-TC Why We Do What We Do: Serologic Testing Event Date: Saturday, October 2, 27 Event Time: 2: PM to 5:3 PM Director Director Lieta Maffei Lieta Maffei CHE, MT(ASCP)SBB CHE, MT(ASCP)SBB 3143 Chauncey Drive 3143 Chauncey Drive San Diego, CA, San Diego, CA, (619) (619) Disclosures: No Disclosures: No Speaker Debra Bailey MT(ASCP)SBB 1 Red Cross Circle Pomona, CA, 91768, USA BaileyDeb@usa.redcross.org Speaker W. John Judd FIBMS, MIBiol 414 Lockwood Lane SW Supply, NC, 28462, USA johnjudd@med.umich.edu Disclosures: No Disclosures: No Speaker Susan Johnson MSTM, MT(ASCP)SBB sue.johnson@bcw.edu Disclosures: No

4 Why do we do what we do? Antibody Detection Antibody detection Context of presentation U.S. practices Pretransfusion testing Debra Bailey MT(ASCP)SBB IRL Assistant Director American Red Cross Blood Services Southern California Region Why do antibody detection? Detect potentially clinically significant irregular RBC antibodies in a potential transfusion recipient.5-2% of the general patient population have irregular RBC antibodies 6-35% of various multiply transfused populations have irregular antibodies Issitt PD, Anstee DJ. Applied Blood Group Serology, 4 th edition 1998 Why do antibody detection? Avoid Transfusion of incompatible donor RBCs Decreased survival of donor RBCs Clinical effects of Hemolytic Transfusion Reactions (HTRs) Causing morbidity Promote Effective RBC transfusion therapy Timely RBC transfusion Historical perspective Antibody detection evolution 195 s Major (patient plasma/donor RBCs) and minor (patient RBCs/donor plasma) crossmatch the standard reading for direct agglutination with and without albumin Some performed testing with selected RBCs to screen for antibodies 1

5 Antibody detection evolution 196 S IAT crossmatch requirement introduced (3 rd edition AABB Standards, 1962) Brief mention that a screen of patient serum for antibodies against selected RBCs could be an alternative to IAT crossmatch (3 rd and 4 th edition AABB Standards only; then removed) Antibody detection evolution 197 s Requirement for the detection of unexpected antibodies using reagent RBCs introduced (5 th edition AABB Standards, 197) Mid 7 s, reports support use of antibody detection instead of IAT crossmatch for pre-surgical patients (cost/benefit), but standards still required IAT crossmatch Antibody detection vs. IAT crossmatch Antibody detection Can be performed in advance Carefully selected RBCs to include all common antigens Is not inventory dependent Does not sequester units for improbable transfusion IAT crossmatch Direct indication of compatibility Capable of detecting antibodies to low incidence antigens Antibody detection evolution 198 s IAT crossmatch no longer required if antibody screen is negative (11 th edition AABB Standards, 1984) Antibody detection current requirements AABB Standards for Blood Banks and Transfusion Services 24 th edition 26 A test for unexpected antibodies to red cell antigens is required methods of testing shall be those that demonstrate clinically significant antibodies. Antibody detection current requirements AABB Standards for Blood Banks and Transfusion Services 24 th edition 26 They shall include incubation at 37C preceding an antiglobulin test using reagent red cells that are not pooled. 2

6 Antibody detectioncurrent Antibody detection has become primary means to ensure a safe & effective transfusion for general patient population Much emphasis has been placed on appropriate detection of irregular red cell antibodies through analysis of the factors that contribute to a successful antibody detection test Factors that contribute to the antibody detection test No standard requirement for selection Method Enhancement media (applicable to tube method only) Configuration of antibody detection RBC sets Reading phases of agglutination (applicable to tube method only) Autocontrol with antibody detection What are we doing? Source data for charts on subsequent slides U.S. data Maffei LM, et al. Transfusion 1998;38: North American data Shulman IA, et al. Archives of Pathology and Laboratory Medicine 25;9: (First Trimester) North American data Personal communication with Shulman IA. Data from first trimester 27 CAP survey. Complete data pending publication by Downes KA, Shulman IA 27 Regional Southern California data Informal hospital survey; unpublished 8 hospitals queried about antibody detection practices Factors that contribute to the antibody detection test No standard requirement for selection Method Enhancement media (applicable to tube method only) Configuration of antibody detection RBC sets Reading phases of agglutination (applicable to tube method only) Autocontrol with antibody detection Antibody detectionmethod Currently available and acceptable options Tube Gel Solid Phase Red Cell Adherence Liquid Microwell Approx. % of total participants 1 Antibody detectionmethod Manual Testing- National Data Year TUBE GEL OTHER 27 1 st trimester only 3

7 Factors that contribute to the antibody detection test No standard requirement for selection Method Enhancement media (applicable to tube method only) Configuration of antibody detection RBC sets Reading phases of agglutination (applicable to tube method only) Autocontrol with antibody detection Antibody detectionenhancement media If antibody detection is performed by tube test, the following enhancement media options are available None ( saline ) Albumin LISS PEG Approx. % of tube method users Antibody detectionenhancement media Manual Testing- National Data Year LISS ALB PEG NONE 27 1 st trimester only Method and enhancement considerations Balance of sensitivity and specificity Strive to detect clinically significant antibodies while avoiding clinically insignificant antibodies Still no one perfect method or enhancement for the detection of clinically significant RBC antibodies Transfusion Services must choose Factors that contribute to the antibody detection test No standard requirement for selection Method Enhancement media (applicable to tube method only) Configuration of antibody detection RBC sets Reading phases of agglutination (applicable to tube method only) Autocontrol with antibody detection Antibody detectionreagent RBC sets FDA require the following antigens be represented on at least one of the antibody detection RBC suspensions in the set D, C, E, c, e, M, N, S, s, P 1, Le a, Le b, K, k, Fy a, Fy b, Jk a and Jk b The AABB standards require and FDA recommends the use of RBCs that are not pooled for pretransfusion testing 4

8 Antibody detectionreagent RBC sets Reagent RBC set options for pretransfusion testing 2 RBC suspension set 3 RBC suspension set Other Pooled- does not meet AABB requirements for pretransfusion testing 4 RBC suspension set- expresses a low incidence antigen of interest Approx. Percent Antibody detectionreagent RBC sets Manual Testing- National Data Year TWO THREE OTHER 27 1 st trimester only Antibody detectionreagent RBC sets Antigen dose 2 RBC suspension set example Difficult to consistently achieve double dose D C E c e P 1 M N S s Le a Le b K k Fy a Fy b Jk a Jk b RBC suspension set example Usually able to provide a double dose D C E c e P 1 M N S s Le a Le b K k Fy a Fy b Jk a Jk b Individuals that inherit two copies of the same gene at a particular locus, will be homozygous for that gene and their RBCs will express a double dose of the corresponding antigen Individuals that inherit two dissimilar genes at a particular locus, will be heterozygous for those genes and their RBCs will express a single dose of each antigen Dose example Genotype: Fy a /Fy a Phenotype: Fy(a+b-) Result: 1% Fy sites = Fy a antigen (double dose) Genotype: Fy a /Fy b Phenotype: Fy(a+b+) Result: 5% of Fy sites = Fy a antigen (single dose) Antibodies that sometimes exhibit dosage Rh: C, E, c, e MNS: M, N, S, s Duffy: Fy a, Fy b Kidd: Jk a, Jk b Example of anti-jk a exhibiting dosage D C E c e Le a Le b M N S s K k gel Fy a Fy b Jk a Jk b IgG PT 5

9 Dosage studies Shulman IA, et al. Laboratory Medicine. 1985;16:62-64 of 26 examples of anti-jk a did not react with some or all Jk(a+b+) by albumin IAT Shulman IA, et al. Laboratory Medicine 1989;2: of 25 examples of anti-fy a did not react with some Fy(a+b+) but reacted with all Fy(a+b-) Antibody detection RBCs considerations Dose Goal Cost Factors that contribute to the antibody detection test No standard requirement for selection Method Enhancement media (applicable to tube method only) Configuration of antibody detection RBC sets Reading phases of agglutination (applicable to tube method only) Autocontrol with antibody detection Antibody detectionreading for agglutination If antibody detection is performed by tube test, reading for agglutination at the following phases is not required Immediate spin (IS) Room temperature (RT) 37C Indirect Antiglobulin Test (IAT) phase is required Antibody detectionreading for agglutination Antibody detectionreading for agglutination Percent National Data IS RT 37C IAT Percent Regional Data IS RT 37C IAT According to the US data, approximately 7 % of those testing by tube test were performing an IS reading, approximately 5% a RT reading during pre-transfusion antibody detection Why? 6

10 Antibody detection- IS? RT? Antibody specificities typically detected a temperatures below 37C Anti-M Anti-I Anti-N Anti-i Anti-P 1 Anti-IH Anti-Le a Anti-H Anti-Le b Anti-Lu a Antibody detection- IS? RT? Common antibodies not reacting at body temperature (37C) are typically rarely considered capable of causing in vivo red cell destruction of antigen positive transfused RBCs D C E c e P 1 M N S s Le a Le b K k Fy a Fy b Jk a Jk b Antibody detection- IS? RT? Detection of such antibodies in pretransfusion testing can cost time and money Identification of such antibodies have little relevance in transfusion safety IS/RT reading when performing antibody detection is not recommended Antibody detection- 37C reading for agglutination? Newer studies have questioned the necessity of the 37C reading for tube testing There is no requirement to perform a reading for agglutination at 37C Antibody detection tests performed using gel test, SPRCA, or PEG IAT by nature have no 37C reading for agglutination Antibody detectionreading for agglutination Antibody detection- 37C reading for agglutination? Percent National Data IS RT 37C IAT Percent Regional Data IS RT 37C IAT Proponents of the 37C reading cite examples of antibody specificities commonly considered clinically significant that have agglutinated at 37C and not reacted by IAT (e.g., anti-e) They are correct but the more important question is, are these antibodies clinically significant? 7

11 Antibody detection- 37C reading for agglutination? Proponents for omitting the 37C reading cite fewer clinically insignificant reactions detected that would have required time, effort and resources to investigate A few studies have shown no increase in HTRs in the labs that have dropped the 37C reading Factors that contribute to the antibody detection test No standard requirement for selection Method Enhancement media (applicable to tube method only) Configuration of antibody detection RBC sets Reading phases of agglutination (applicable to tube method only) Autocontrol with antibody detection Antibody detectionroutine autocontrol Antibody detectionroutine autocontrol National Data YES NO Other 27 Regional Data 6 94 YES NO Was thought to be needed to detect Antibodies adsorbed onto transfused RBCs Auto or drug induced antibodies Studies have shown that the predictive value of routinely including an autocontrol during antibody detection for detecting clinically significant alloantibodies to be lower than routinely performing an antiglobulin crossmatch for a patient with a negative antibody screen One last thought Finally Information obtained from a positive antibody screen is valuable information to be used as we move into antibody identification Antibody detection RBCs that are non-reactive with the patient s plasma can be used just like panel RBCs to exclude red cell antibodies Strength and phase of reactivity (if applicable) of reactive RBCs can be clues to streamline the identification process Why do you do what you do???? It is a good idea from time to time to evaluate our testing practices to determine if the reasons why we are doing what we are doing are still applicable 8

12 Literature Literature Boral LI, et al. The type and screen: a safe alternative and supplement in selected surgical procedures. 1977;17: Treacy M. Pretransfusion testing for the 8s: a technical workshop. AABB 198. Garratty, G. The role of compatibility tests. Transfusion 1982;22: Shulman IA, et al. The dependence of anti-jka detection on screening cell zygosity. Laboratory Medicine 1985;16: Judd WJ, et al. The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited. Transfusion 1986;26: Stec N, et al. The efficacy of performing red cell elution studies in the pretransfusion testing of patients with positive direct antiglobulin tests. Transfusion 1986;26: Shulman IA, et al. Influence of reagent red cell zygosity on anti-fya detection. Laboratory Medicine 1989;2: Literature Literature Pestaner JP, et al. Is it safe to omit the 37C reading from pretransfusion red blood cell antibody detection testing? American Journal of Clinical Pathology 1994;11: Beck, ML, et al. Red cell compatibility testing: a perspective for the future. Transfusion Medicine Reviews. 1996;X: Judd WJ. Commentary: testing for unexpected red cell antibodies-two or three reagent red cell samples. Immunohematology 1997;13:9-92. Issit PD, Anstee DJ. Applied Blood Group Serology, 4th edition. Durham, NC: Montgomery Scientific Publications Maffei LM, et al. Survey on pretransfusion testing. Transfusion 1998;38: Issitt PD. Antibody screening: elimination of another piece of the test. Transfusion 1999;39: Judd WJ, et al. Revisiting the issue: can the reading for serologic reactivity following 37C incubation be omitted? Transfusion 1999;39: Judd WJ. Modern approaches to pretransfusion testing. Immunohematology 1999;15: Literature Literature Issitt PD. From kill to overkill: 1 years of (perhaps too much) progress. Immunohematology 2;16: Shulman IA, et al. Pretransfusion compatibility testing for red blood cell administration. Current Opinion in Hematology 21;8: Garratty G. Screening for RBC antibodies-what should we expect from antibody detection RBCs. Immunohematology 22;18:71-77 Duran J, et al. Antibody screening in 37C saline. Is it safe to omit it using the indirect antiglobulin (gel) test? Immunohematology 22;18:13-15 Shulman IA, et al. North American pretransfusion testing practices, Archives of Pathology and Laboratory Medicine 25;9: Brecher ME (ed). Technical Manual, 15th edition. AABB. Bethesda, MD 25. Standards for Blood Banks and Transfusion Services, 24th edition. AABB Bethesda, MD 26 Casina TS. In search of the Holy Grail: comparison of antibody screening methods. Immunohematology 26;22:

13 What is a Crossmatch? Why We Do What We Do: Crossmatch A test performed to determine the compatibility of a donated unit of blood for its intended recipient Terry Downs MT(ASCP)SBB Supervisor University of Michigan Health System tdowns@umich.edu Why do we Crossmatch? Crossmatch Methods Determine whether the recipient serum contains an antibody capability of reacting with and destroying the donor red cells. This includes ABO antibodies. Tube Gel column Microtiter Computer What We Used to Do Currently We performed an antiglobulin crossmatch on everyone-is, 37 C and IAT If we didn t, we made the physician come to the Blood Bank to sign the consent to transfuse uncrossmatched blood Computer Immediate Spin Antiglobulin No crossmatch at all! 1

14 What do the Standards Say? Standards, continued Before issue, a sample of the recipient s serum shall be crossmatched against a sample of donor cells from an integrally attached red blood cell segment The crossmatch shall use methods that demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens and shall include an antiglobulin test If no clinically significant antibodies were detected in tests and there is no record of previous detection of such antibodies, detection of ABO incompatibility shall be performed AABB Standards, 24 th Edition AABB Standards, 24 th Edition Standards, continued Standards, continued Computer Crossmatch Validated on site 2 determinations of ABO are made: Current sample Historical or 2 nd on current sample Computer contains unit number, component name, ABO, Rh, confirmed ABO Computer crossmatch: Method exists to verify correct entry before release of components System contains logic to alert user to ABO/Rh discrepancies between the donor and the recipient AABB Standards, 24 th Edition What About the FDA? FDA 21 CFR : Method of collecting and identifying the blood samples of recipients to ensure positive ID Use of fresh recipient serum or plasma less than 3 days old for all pretransfusion testing if the recipient has been pregnant or transfused Procedures to demonstrate incompatibility between the donor s cell type and the recipient s serum or plasma type A provision that, if the unit of donor s blood has not been screened by a method that will demonstrate agglutinating, coating and hemolytic antibodies, the recipient s cells shall be tested with the donor s serum (minor crossmatch) Procedures to expedite transfusion in lifethreatening emergencies (signed by a physician) 2

15 Antiglobulin Crossmatch Other Methods Use when patient has antibodies to clinically significant antigens Use when immediate spin crossmatch is positive Patient has clinically insignificant room temperature antibody No clinically significant antibodies in sample No history of clinically significant antibodies Method to detect ABO incompatibility Immediate spin Computer Immediate Spin Crossmatch Advantages of IS XM Works well for Blood Banks that do not have the computer system needed for computer crossmatches Comfort level when performing a serological test Decreased turn around time versus antiglobulin crossmatch Decreased workload Reduced reagent costs Effective use of blood inventory Computer Crossmatch Advantages of Computer XM Even faster than immediate spin, but Validate on site System contains logic to alert the user to discrepancies 2 ABO determinations Current sample Historical sample or 2 nd on current sample Decreased turnaround time Decreased workload Reduced sample volume for pretransfusion testing Reduced exposure to personnel Better use of blood inventory 3

16 What Kind of Crossmatch to Perform? History of Clinically Significant Antibodies? Depends on SOPs Depends on comfort level of Medical Director and staff Need guidelines in place when various crossmatches are in SOP Depends on the patient s results and urgency of the blood No Current Antibody Screen Negative? Yes Immediate Spin or Computer What About Patients with Antibodies? What Determines Clinical Significance? Depends on the clinical significance of the antibody Is the antibody known to cause destruction of red cells? Antibodies reactive at 37 C and/or IAT are generally significant Reactivity at room temp and below are generally not significant There are always exceptions! Medical Director to set policy Is the antibody clinically significant? When NOT to perform the antiglobulin crossmatch Yes Perform Antiglobulin Crossmatch Yes Perform Antiglobulin Crossmatch No Antibody Screen Positive? No Perform Immediate Spin or Computer Crossmatch (Immediate Spin may be positive) Massive transfusions-replace blood volume in 24 hours Pretransfusion serum does not represent the blood currently in the patient Only required to confirm ABO compatibility Can use immediate spin or computer 4

17 Massive Transfusion in Patients with Antibodies Antibody usually not present to cause problems at that point After stabilization, resume antiglobulin crossmatch Note to physician of potential for delayed reaction When NOT to Crossmatch? Infants Emergencies Infants Infants Difficult to obtain blood samples Neonates do not typically form antibodies during the first 4 months of life Use Group O reds cells for transfusion Check for passive antibodies if type specific red cells are used If infant has passive antibody from mother, issue antigen negative red cells Continue for as long as maternal antibody persists in infant Crossmatch not necessary when using Group O red cells Emergencies Resources Patient bleeding-any blood may better than no blood at all Issue Group O red cells for emergencies Not the time for staff to consider options Important to have procedures in place to define what blood to give Crossmatch after the fact when calmer AABB Technical Manual, 15 th Edition AABB Standards for Blood Banks and Transfusion Services, 24 th Edition Code of Federal Regulations 5

18 Why Do We Do What We Do? ABO TYPING AABB Annual Meeting 27 Susan T. Johnson, MSTM, MT(ASCP)SBB BloodCenter of Wisconsin Milwaukee, WI ABO TYPING Why do we ABO type? What are we detecting? Why do anti-a & anti-b cause hemolysis? Should we worry about anti-a 1? LANDSTEINER S RULE Why is ABO important? The plasma of an individual contains naturally occurring antibodies to A and B antigens, if these antigens are absent from the individual s red cells. ACUTE IMMUNE HEMOLYSIS ABO antibodies cause the most serious form of acute immune hemolysis Intravascular Fatality Rate of 2% 1

19 WHY DO ANTI-A & ANTI-B CAUSE HEMOLYSIS? PATHOGENECITY OF ABO ANTIBODIES Antigen Characteristics Antibody Characteristics ANTIGEN CHARACTERISTICS Antigenic Make-up ABO ANTIGEN QUANTITY Carbohydrate antigen Quantity Distribution Up to 1.5 million antigens per RBC! The Sugars Numbering the Carbons HO HO HO HO HO D-Galactose HO HO O HO N HO N-Acetylglucosamine 6 HO 6 HO HO 5 1 HO 5 1 O HO 3 HO 3 HO N HO HO D-Galactose N-Acetylglucosamine 2

20 GlcNAC GlcNAC i ANTIGEN Type II Unbranched Chain Gal GlcNAC R GlcNAC Gal I ANTIGEN Gal β-1,6-acetylglucosaminyltransferase I gene Gal Gal β-1,6-acetylglucosaminyltransferase Branching Enzyme GlcNAC Gal GlcNAC Gal GlcNAC ABH GLYCOSYLTRANSFERASES H ANTIGEN GENE H (FUT 1) A 1 GENE PRODUCT α -2-L-fucosyltransferase α -3-N-acetylgalactosaminyltransferase H gene α -2-L-fucosyltransferase GlcNAC Gal B α -3-D-galactosyltransferase Gal O None 3

21 A gene A ANTIGEN α -3-N-acetylgalactosaminyltransferase Gal B gene α -3-D-galactosyltransferase B ANTIGEN Gal Gal GlcNAC Gal GlcNAC GalNAC Gal H 1 to H 3 BRANCHING Gal - GlcNAc - Gal R Gal - GlcNAc - Gal - GlcNAc - Gal - R H 1 (i) H 2 (I) Gal - GlcNAc Gal - GlcNAc - Gal - GlcNAc - Gal R H 3 H 4 Gal - GlcNAc Gal - GlcNAc Gal - GlcNAc (Gal GlcNAc) n -Gal -Glc-R H 4 Gal - GlcNAc 4

22 ABH ANTIGENS AT BIRTH i I H Aor B Weaker expression Occasionally mixed field Branching enzyme not active Lack branched H 3 & H 4 glycolipids (I-i+) As i converts to I; H, A, B ABH ANTIGEN DISTRIBUTION Carried on protein and lipid structures Most are glycoproteins carried on. Anion Transport Protein (Band 3) 1,, copies Glucose Transport Protein (Band 4.5) 5, copies Rh Associated Glycoprotein (RhAG) 5-1% associated with glycolipids GLYCOLIPID C/c HISTO-BLOOD GROUP ANTIGENS Skin Vascular endothelium Digestive epithelia Colon Small intestine Respiratory mucosae - Lung Most Epithelial Cells Platelets Low levels on most ABO ANTIGENS Environment Bacteria Animals 5

23 ANTIBODY CHARACTERISTICS Factors Influencing Antibody Pathogenicity Antibody characteristics: Class (IgM vs IgG) Subclass Specificity Thermal Range Efficiency of complement activation Antigen affinity Adapted from Petz and Garratty Immune Hemolytic Anemia, 2 nd ed. 24 Factors Influencing Antibody Pathogenicity Quantity of RBC-bound antibody and/or complement Type of complement on RBCs Activity of RE system ABO ANTIBODIES Non-red cell stimulated ( naturally occuring ) IgM / IgG detected at IS, RT, 4C Active at 37C Present in high titer Efficient activator of complement Cause intravascular hemolysis Adapted from Petz and Garratty Immune Hemolytic Anemia, 2 nd ed. 24 Intravascular Hemolysis ABO TYPING B RBC IgG antibody C1 Complement recognition unit C2aC3C4b Complement activation unit C5b6789 Membrane attack complex RBC l ysis 6

24 BARNEY I ve got Bs in my blood! Anti-A 1 IS ANTI-A 1 SIGNIFICANT? Found in A 2, A 2 B and weaker subgroups of A A 1 AND A 2 PHENOTYPES Qualitative difference A 2 can make anti-a 1 Transferase activity differs A 1 transferase adds GalNAc to repetitive Type 3H and Type 4H Type 2A Type 2H GalNAc-Gal-GlcNAc 7

25 Repetitive Type 3H Structures Type 3A Repetitive Type 3A Structures Type 3H Type 3H Type 2A Type 2A Type 2H Type 2H Gal - GalNAc-Gal-GlcNAc GalNAc - Gal - GalNAc-Gal-GlcNAc Present on glycolipids SIGNIFICANCE OF ANTI-A 1 Rarely causes hemolysis 4 reports in the literature of HTR 1946, 1959, 1975, 1978 Antibody must be reactive at 37C ABO TYPING - SUMMARY Why do we ABO type? What are we detecting? Why do anti-a & anti-b cause hemolysis? Should we worry about anti-a 1? 8

26 What to do about Weak D? W. John Judd, FIBMS, MIBiol April, 27 University of Michigan, Ann Arbor Objectives Understand the fundamental difference between Weak D and Partial D phenotypes Develop a testing strategy to decrease RhD alloimmunization in Partial D patients Assess the impact of such a strategy on the supply of Rh-negative blood when applied to transfusion candidates What is D? multi-pass membrane protein encoded by RHD at 1p36.13-p34 its presence on RBCs defines the Rhpositive phenotype highly immunogenic, second only in clinical importance to A and B antigens What is D? first defined by an agglutinin (IgM) in the serum of Mary Seno, who delivered a stillborn fetus (Levine and Stetson, 1939) initially thought to be the same determinant as that defined by diluted antibody from rabbits/guinea pigs immunized with Rhesus monkey cells (Landsteiner and Wiener, 194) What is D? upstream Rh box RHD downstream Rh box RHCE NH 2 COOH SMP1 1

27 CE polypeptide Cc Ee Rh-positive Rh-negative NH 2 COOH hybrid Rh box Molecular Backgrounds for D- Distribution of D Antigen English RHD deletion D- D-C-E- African 18% RHD deletion 67% RHDψ (37-bp insert; TYR 259 stop) 15% RHD-CE-D S (r S ; VS+, V-) Japanese 63% RHD deletion 28% (all C+) intact RHD 2% RHD-CE-D English Nigerian Chinese SE Michigan Data %C %AA %C %AA R 1 r R O R 1 R R 1 R Z.1 - R 1 R rr R 2 r r r R 2 R r r.4.1 The Entity Known as D u 1 st described by Stratton* in 1946 mistakenly called the D u antigen results from: Cde haplotype in trans (Ceppellini effect) weak D alleles hybrid Rh genes * Developed Stratton sandwich technique for Rh D typing. 2

28 The Ceppellini Effect Weak D Phenotypes Flegel and Wagner: Clin Lab 22: D u DCe/Ce ce/ce Ce/ce D u Ce/ce DCe/ce Ce/ce DCe/Ce DCe/ce ce/ce DCe/ce arise from missense mutations to regions of RHD encoding transmembrane portion of D less D protein inserted into RBC membrane usually do not make anti-d if exposed to normal Rh+ RBCs should type as Rh+ with high-affinity IgM monoclonal anti-d partial D including D categories weak D types Partial D Phenotypes arise from hybrid genes and missense mutations to regions of RHD encoding parts of D external to RBC membrane some react weakly with anti-d may make antibody to missing part of D if transfused, should receive Rh- negative RBCs if pregnant, are candidates for RhIG account for FDA-reportable errors when hospitals and blood centers use different anti-d reagents 5 RHCE 3 IIIa IIIb V11 DFR IVa r G RHD 3 IVb Va V1.1 r G D-V-VS+ R O Har 3

29 partial D novel sequence Reasons to Test for Weak D conserve Rh-negative blood for true D- negative recipients* do early in pregnancy, to eliminate falsepositive results due to large FMH at delivery avoid giving RhIG to women who do not need it* NH 2 COOH * Arguments viable only if weak D individuals cannot make anti-d. Frequency of Weak D If You Don t Test for Weak D Hopkins Scotland % if frequency of weak D is.4%, and Garretta Beck* France USA %.2% if frequency of D-negatives is 16.% frequency of weak D among apparent D-negatives is (.4 16) x 1 = 2.5% Jenkins USA 24.4% 2.5% RhIG doses go to weak D women Flegel Germany 24.4% 2.5% Rh-negative RBCs go to weak D recipients * Frequency of DVI =.2%. Should We Test for Weak D? mab Anti-D s required for donor blood IgM IgG not required for recipients tube Ortho MAD2 human standard-of-care for infants of Rhwomen (to determine need for RhIG) not required for prenatal testing tube tube tube Gamma Immucor-4 Immucor-5 GAMA 41 MS21 Th28 F8D8 MS26 MS26 gel ID-MTS MS21 n/a 4

30 Direct Tests (IS) Direct Tests DII DIIIa* DIIIb DIVa DIVb G-T 1 1 mf I 4 -T mf 9 I 5 -T 11 mf 8 1 * transfused with Rh- RBCs O-T 11 mf 8 O-G mf 11 DVI.1 DVI.2 DFR G-T I 4 -T I 5 -T O-T tube tests by IS and after 37 C incubation O-G Indirect Tests (IAT) Conflicts in Direct Tests G-T I 4 -T I 5 -T O-T O-G DVI.1 DVI.2 DFR G-T 11 I 4 -T 11 1 I 5 -T 11 1 O-T DVa DBT R o Har-1 R o Har (8) 6(6) 9 11 (6) 6(1) 8 1 () 6(1) () () R o Har-3 3(4) (3) (3) () 9 ( )= after incubation Findings at IAT We Switched to DVa DBT R o Har-1 R o Har-2 R o Har-3 G-T 1 / I 4 -T / I 5 -T / O-T normal RHD 1 corrupted intron 4/5 2 weak D Type 1 6 weak D Type 2 3 DAR (weak D Type 4.2) DVI by exon mapping DVa by serology (mab panel) 5

31 Strategies for Partial D Pretransfusion/Prenatal Testing do not test for weak D give Rh-negative RBCs; give RhIG consider two anti-d s; look for conflicts grade reactions; interpret weak reactors as Rh-negative Partial D in Pregnancy partial D women at risk most mab anti-d do not detect D VI (RhIG presumed effective) most mab anti-d react strongly with R O Har RBCs in contrast to human anti-d; R O Har women unlikely to receive RhIG if tested with modern-day reagents infant Rh+ no no RhIG direct test with anti-d yes no >3+ give RhIG at weeks rosette test yes yes no RhIG no single dose of RhIG evaluate by K-B Strategies for Partial D Donor Testing* select reagents test for weak D (tube IAT) or manage the conflicts * to avoid FDA-reportable errors Terminology Issues many partial D s react weakly with anti-d some weak D s make anti-d consider all as D-variants with clear indication of those that can produce anti-d Food For Thought Why is prevention of anti-d formation so sacrosanct? Should we not provide CcEeKmatched RBCs for all (female age <5) patients? 6