Webinar: Association of Hgb A Clearance & RBC Antibodies
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1 Webinar: Association of Hgb A Clearance & RBC Antibodies
2 Second Webinar Session A second session of this webinar will be hosted Wednesday, July 12 2:00 PM EST (1800 GMT) Register at the link below: All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
3 Handouts us/pages/educational Program Handouts.aspx All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
4 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is July 28, 2017 Certificates will be sent via only to those who have registered by August 11, 2017 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
5 Presentation Recording Session will be recorded and posted. Access information will be sent to each registrant when the recording becomes available No CE issued for participating in recording All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
6 Questions? You are all muted Q&A following session Type in questions All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
7 Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations. The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor. All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
8 Donor Hemoglobin A Clearance and RBC Antigen Genotyping in Chronically Transfused Children with Sickle Cell Anemia Marianne E. McPherson Yee, MD Assistant Professor of Pediatrics Emory University School of Medicine Clinical Director of Sickle Cell Disease Program, Children s Healthcare of Atlanta at Egleston Hospital Atlanta, GA
9 Objectives Review Chronic Transfusion Therapy (CTT) for sickle cell anemia (SCA) Alloimmunization prevention strategies Role of HEA in transfusion matching Present a prospective study of RBC genotyping of SCA patients and transfused units during CTT Antigen frequencies, antigen mismatch frequencies Alloimmunization events Variables effecting Donor HbA clearance Clinical Applications
10 Chronic Transfusion Therapy in SCA Primary therapy for stroke prevention in SCA Majority of SCA patients will receive intermittent transfusions during childhood 10% pediatric SCA patients will receive CTT
11 Transfusion in SCA Alloimmunization the major barrier to transfusion therapy in SCA. Prevalence: 18 40% or higher, despite serologic antigen matching Alloimmunized patients are more likely to have future alloimmunization events Consequences Hemolytic Transfusion reactions Limits availability of compatible RBC units.
12 Antigen Matching in SCA Strategies to minimize alloimmunization: Limited matching* (no prior alloimmunization) Extended matching (past alloimmunization) ABO, D C/c, E/e, K ABO, D C/c, E/e, K, Fy a, Jk b, + antigens to which patient is alloimmunized Additional Transfusion Requirements in SCA: Sickle negative; leukoreduced Age <21 days, if possible * NHLBI Evidence Based Management of SCD: Expert Panel Report, 2014
13 Limitations of serologic matching in SCA Lack of anti sera for many antigens Inability to identify genetic variants, particularly RH Interference from recent transfusions
14 RH genetic variants in SCA Serologic typing does not identify RH variants Study of multiply transfused SCA patients (Chou et al. Blood 2013): Majority had Rh antibodies. Unexplained Rh antibodies occurred in patients who were phenotypically (+) for that antigen RH genotyping (RHCE, RHD BeadChip arrays): 87% of patients had variant alleles ( 1 conventional allele) Failure to recognize RH variants in patients and donors may lead to alloimmunization and DHTR. Immucor RHCE, RHD and HEA LR BeadChip assays are available as Research Use Only in the US.
15 Chronic Transfusion Therapy in SCA How does CTT work? 1. Dilutes sickle RBC with normal (HbA) RBC 2. Raises hemoglobin, therefore suppresses erythropoiesis, When does CTT not work well? a) Hemolytic transfusion reaction occurs b) Pre transfusion Hb low, HbS high. Transfused RBC being cleared too rapidly c) Pre transfusion Hb high, HbS high Erythorpoiesis not adequately suppressed
16 Survival of transfused RBC in vivo Donor (unit) Factors Age of RBC units Donor genetics G6PD Donor/Recipient Interaction Antigen Mismatches Antibody mediated destruction (DHTR) Non humoral immunologic clearance? Recipient Factors Age of patient Alloimmunization Spleen status Complement activation
17 SCA Antigen Mismatch Study Observational study of RBC antigen mismatches in pediatric SCA patients during CTT Aims: Compare RBC antigen frequencies in SCA patients vs. donor units. Determine the frequency of minor antigen mismatches per transfusion episode Observe antigen exposures prior to alloimmunization events Determine if clearance of transfused RBC (HbA RBC) is associated with antigen mismatches
18 Study Design & Methods Prospective study Enrolled patients followed for 12 months each 2 institutions with 4 blood bank locations: Children s Healthcare of Atlanta Children s National Health System Standard of care transfusion practices: Sickle negative, leukoreduced RBC Antigen matching: Category 1: Non alloimmunized: C/c, E/e, K Category 2: Alloimmunized: C/c, E/e, K, Fy a, Jk b, alloabs
19 Study Methods: RBC genotyping Patient Testing PreciseType HEA RHCE and RHD BeadChip assay Leukoreduced RBC Unit Testing HEA LR Prototype RHCE and RHD genotyping not feasible (except possible partial C (r S ))
20 PreciseType HEA
21 PreciseType HEA ISBT Blood Group Antigens Predicted by HEA with licensed anti sera Antigens Predicted by HEA without licensed anti sera RhCE C, c, E, e V, VS (possible r S partial C) Kell K, k Kp a, Kp b, Js a, Js b Duffy Fy a, Fy b Fy(a b )(GATA 67C) Kidd Jk a, Jk b MNS M, N, S, s U Lutheran Lu b Lu a Diego Colton Dombrock LW Sciana Di a, Di b Co a, Co b Do a,do b, Hy, Jo a Lw a, LW b SC1, SC2
22 HEA LR BeadChip Leukoreduced (LR) RBC Unit: 5 x 10 6 WBC/unit Extract DNA from 3 4 unit segments Same panel of SNPs as PreciseType HEA Assay Does not assess SNPs associated with RHCE, RHD variants
23 RHCE and RHD BeadChip Assays Identifies >75 RHD and >35 RHCE variants
24 Study Design Hb A (g/dl) Patient 1 To 12 mo Hb S (g/dl) Months
25 HbA Estimation HbA = (HbA Post Txn) (HbAPre next Txn) ( # days btwn Txn) Hb A (g/dl) days Post txn HbA was directly measured in ~20% of transfusions Post txn HbA was estimated from unit volume, unit Hct, patient weight in remaining ~80% transfusions (estimate had high accuracy with bias 0.33 g/dl).
26 Patient Characteristics 90 pediatric patients (88 African American, 2 Hispanic) Mean age 11.4 yrs ( yrs) 1,134 transfusion episodes (2,320 LR RBC units) At Entry At Exit Category 1 Matching Non Responders Category 2 Matching Responders Category 1 2 Switching 1 patient with new Ab of undetermined specificity (AUS) 1 with historic anti e, found to have hr B variant (ces/ces)
27 Patient Antibody Histories Blood Group Alloantibodies Frequency at Entry Frequency at Exit RH C, E, e, Go a, f, V, VS, C w (1 anti e, 1 anti Go a ) KELL K, Kp a, Js a 9 12 (3 anti Js a ) Duffy Fy a, Fy3 2 2 Kidd Jk b 1 1 MNS M, S 4 4 Lutheran Lu a 3 3 Knops Knops 1 1 Lewis Le b 1 1 Other AUS, Wr a 0 2 (1 anti Wr a, 1 AUS) TOTALS 41 48
28 Patient RH Genotypes Conventional / Conventional Conventional / Variant Variant / Variant Compound heterozygotes (e.g. RHCE ce(733g)/cemo) Homozygous variants (e.g. RHCE ce(48c)/ce(48c)) Not considered to be at risk for alloimmunization to the conventional Rh antigens expressed Considered to have an antigen mismatch when transfused with antigenpositive units
29 RHCE Patient Genotypes RHCE Conventional / Conventional 21 (23.3%) Conventional / Variant 37 (41.1%) Variant expression only* Partial e Partial e and C (ce48c/ce(48c,733g,1006t) Partial C (ce/ce(48c,733g,1006t) Partial c (Ce/ceTI type 1) Possible Partial e (ce(48c) and ce(733g), unknown cis or trans) (36.7%) Potential for mismatches when transfused with antigen positive units
30 RHD Patient Genotypes RHD Conventional / Conventional (RHD+) 63 (70.0%) Conventional / Variant (RHD+) 18 (20.0%) Variant expression only Partial RHD+ Partial RHD Weak D+ RHD Deletion / RHD Deletion Potential for 4 mismatches when 1 transfused with RHD+ 1 units 3 9 (10.0%)
31 2320 RBC units (1135 transfusions) 1470 Category Category units with HEA 1293 Category Category units without HEA 89 HEA LR failure * 196 not tested * Due to inadequate DNA concentration 143 units in a transfusion episode with 1 missing HEA Transfusions evaluated for antigen mismatches 1892 units (932 transfusions) Transfusions evaluated for HbA clearance 622 Transfusions With reliable ΔHbA Estimates 390 Category Category 1
32 Antigen Frequencies: Patients vs. Units * * * * Significant patient vs. unit differences in Ag frequencies: Fya (11% vs. 24%), S (23% vs 38%), U (95.6 vs 99.7%) conventional C (21% vs 13%)
33 Antigen Frequencies Antigen frequencies are similar between patients and donor group for most antigens in HEA panel Does this translate to few mismatches per transfusion episode?
34 Antigen mismatches per transfusion % % % Frequency % 13.2% 6.0% % 3.4% 0.01% 0.006% Antigen Mismatches per Transfusion
35 Antigen Mismatches Minimum # antigens matched Category 1 Transfusions 3 (C/c, E/e, K) Category 2 Transfusions 5 (C/c, E/e, K, Fya, Jkb, alloabs) Mean # of antigen Mismatches Fewer mismatches in Category 1 than would be expected May be due to selection of ethnically similar donors and/or donors who are phenotypically similar for C, E, K.
36 Antigen mismatches per transfusion 932 transfusions (627 cat 1, 305 cat 2) = 1890 units Excluding potential RHCE and RHD mismatches, due to lack of unit genotypes
37 Antigen mismatches per transfusion * * * Matched for Cat transfusions (627 cat 1, 305 cat 2) = 1890 units Excluding potential RHCE and RHD mismatches, due to lack of unit genotypes
38 Alloimmunization Events 7 new alloantibodies in 5 patients Subject ID Category Prior Alloantibodies CTT Duration (years) NEW ANTIBODY S E 6.9 Jsa E S,Jkb, WAA 1.8 Go(a) E AUS, Kpa, C 8 Jsa E K, E, S, Lua, WAA, AUS 10 Jsa Wra H none 6.1 AUS Incidence of Alloimmunization 0.706/100 units (Category 2) 0.068/100 units (Category 1) p=0.02
39 Alloimmunization Events 7 new alloantibodies in 5 patients Subject ID Category Prior Alloantibodies CTT Duration (years) NEW ANTIBODY S E 6.9 Jsa E S,Jkb, WAA 1.8 Go(a) E AUS, Kpa, C 8 Jsa E K, E, S, Lua, WAA, AUS 10 Jsa Wra H none 6.1 AUS Incidence of Alloimmunization 0.706/100 units (Category 2) 0.068/100 units (Category 1) p=0.02 Demonstrates increased frequency of alloimmunization in SCA patients with prior alloimmunization
40 Js a Antigen Kell blood group Antigen Frequency African American: ~20% Caucasian <0.01% ON SCA CTT Study: Patients 18% Units 9% Mismatch: 13.3% Frequency Patients Units Mismatch Js a Antigen Anti sera for Js a is not readily available
41 Js a exposures 74 (82%) Js a ( ) patients N=49 Category 1 N=25 Category 2 22 Jsa( ) patients 3 with anti Jsa 1168 units transfused 101/1014 Jsa(+) 9.96 exposures/100 units p= units transfused prior to anti Jsa 52/546 Jsa(+) 9.52 exposures/100 units 3 new anti Js a 20.4 exposures/ 100 units p= no anti Js a 8.33 exposures/ 100 units
42 Js a Alloimmunization Associated with significantly higher frequency of Jsa exposure (20.4/100 units) vs. patients without Jsa alloimmunization (8.3/100 units) Illustrates that pattern of alloimmunization is shifting rather than being eliminated with standard antigen matching practices. By providing ethnically similar blood to SCA patients, alloimmunization is occurring to low frequency antigens that are more common in African origin populations Examples: Jsa, Goa, f, V, VS
43 Clearance of donor RBC No delayed hemolytic transfusion reactions (DHTR) during the study period Exploratory Aim of study: Determine if antigen mismatches (in the absence of detectable RBC antibody) are associated with increased clearance of transfused RBC (HbA RBC)
44 HbA variation 0.25 ΔHbA per transfusion (g/dl/day) Mean ΔHbA = g/dl Individual Transfusion Episodes
45 ΔHbA variation p= g/dl/day = 2.30 g/dl/28 days g/dl/day = 2.50 g/dl/28 days
46 Category 1 vs. Category 2 Why would a higher level of antigen matching be associated with more rapid clearance of HbA RBC? Hypothesize: Patient with past Alloimmunization (or autoantibodies) Receives Category 2 Transfusion matching Patient is Immunologic Responder to RBC antigens Donor RBC (HbA RBC) are cleared more rapidly (possibly via non humoral immune mechanism)
47 Category 1 vs. 2 Patients Category 1 (Non responders) Category 2 (Responders) Age (years), mean (SD) 10.7 (4.0) 12.5 (3.6) p Volume (ml/kg), mean (SD) 12.9 (2.8) 13.1 (2.7) 0.23 Pre txn Hb (g/dl) 9.65 (0.84) 9.39 (0.88) 0.17 Pre txn HbS (%) 24.3 (11.9) 23.8 (10.4) 0.81 Mean age of units (days) 16.8 (6.7) 16.4 (6.3) 0.47 *limited to transfusion episodes with interval 45 days
48 HbA Clearance Associated with Responder status to RBC antigens Other factors explored: Variable Effect on HbA Age of RBC units Not significant Low G6PD level of units Increased clearance Total # of antigens mismatched Not significant Specific antigen or blood group mismatches
49 HbA Clearance (ΔHbA) Category 2 transfusions (n=234): ΔHbA vs. Blood Group mismatches p RH KELL KIDD DUFFY n/a MNS LUTH DIEGO n/a DOMBROCK COLTON LW SCIANA n/a Note: Category 2 = matched for Jk b (not routinely for Jk a ) * In few instances, Jk a matching could not be honored, due to limited blood availability when matching for rare antigens (e.g. e variants)
50 HbA and Kidd mismatches Category 1 transfusions KIDD mismatches No KIDD mismatch p ΔHbA g/dl/day, Median Category 2 transfusions KIDD mismatches No KIDD mismatch p ΔHbA g/dl/day, Median (95% CI) (0.093, 0.114) (0.083, 0.092) 0.008
51 HbA and Kidd mismatches Category 1 transfusions KIDD mismatches No KIDD mismatch p ΔHbA g/dl/day, Median Category 2 transfusions KIDD mismatches No KIDD mismatch p ΔHbA g/dl/day, Median (95% CI) (0.093, 0.114) (0.083, 0.092) While Kidd mismatches were associated with higher HbA, this has not yet been validated in repeat studies at CHOA/CNMC or elsewhere
52 Conclusions Clearance of donor RBC (HbA RBC) following a transfusion episode is variable, with numerous potential influences: Immunologic Responder status of the patient (alloimmunized vs. non alloimmunized) Specific antigen mismatches may mediate increased clearance, even in absence of alloimmunization or detectable antibodies
53 Clinical Applications RBC antigen typing in SCA patients (CHOA/CNMC) Historically: Serologic phenotype obtained in all SCA patients Contemporary: HEA genotype obtained in all SCA patients (including those with serologic phenotyping)
54 Clinical Applications To date, Antigen matching protocols remain unchanged Category 1: C/c, E/e, K Category 2: C/c, E/e, K, Fya, Jkb, alloantibodies Should there be consideration of matching for low frequency antigens that are more common in Africanorigin donors (Js a, V, VS, etc) in at risk SCA patients? This approach could be made feasible by HEA typing of patients & donors
55 Clinical Applications Supports further investigation of factors that influence clearance of transfused RBC after transfusion, in order to optimize transfusional support for patients with SCA
56 Questions? You are all muted Q&A following session Type in questions All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
57 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is July 28, 2017 Certificates will be sent via only to those who have registered by August 11, 2017 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
58 Questions? You are all muted Q&A following session Type in questions All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
59 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.
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