webinar: association of hgb a clearance & rbc antibodies program handouts... · aflac cancer...

Webinar: Association of Hgb A Clearance & RBC Antibodies

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A second session of this webinar will be hosted

Wednesday, July 122:00 PM EST(1800 GMT)

Register at the link below:https://attendee.gotowebinar.com/rt/9012031991808089089


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http://www.immucor.com/en‐us/Pages/Educational‐Program‐Handouts.aspx


Continuing Education

• PACE, Florida and California DHS• 1.0 Contact Hours• Each attendee must register to receive CE at:

https://www.surveymonkey.com/r/12July2017Webinar

• Registration deadline is July 28, 2017

• Certificates will be sent via email only to those who have registered by August 11, 2017


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• No CE issued for participating in recording


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• 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.

Donor Hemoglobin A Clearance and RBC Antigen Genotyping in Chronically Transfused Children withSickle Cell Anemia

Marianne E. McPherson Yee, MDAssistant Professor of PediatricsEmory University School of MedicineClinical Director of Sickle Cell Disease Program, Children’s Healthcare of Atlanta at Egleston HospitalAtlanta, GA

Aflac Cancer and Blood Disorders Center | Emory University

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


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


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.


Antigen Matching in SCA Strategies to minimize alloimmunization:

Additional Transfusion Requirements in SCA: Sickle‐negative; leukoreduced Age <21 days, if possible

* NHLBI Evidence‐Based Management of SCD: Expert Panel Report, 2014

Limited matching*(no prior alloimmunization)

ABO, DC/c, E/e, K

Extended matching(past alloimmunization)

ABO, DC/c, E/e, K, Fya, Jkb, + antigens to which patient is alloimmunized


Limitations of serologic matching in SCA

Lack of anti‐sera for many antigens Inability to identify genetic variants, particularly RH Interference from recent transfusions


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.


Chronic Transfusion Therapy in SCA How does CTT work?

1. Dilutes sickle‐RBC with normal (HbA)‐RBC2. Raises hemoglobin, therefore suppresses erythropoiesis,

When does CTT not work well?a) Hemolytic transfusion reaction occursb) Pre‐transfusion Hb low, HbS high. Transfused RBC being cleared too rapidly

c) Pre‐transfusion Hb high, HbS high Erythorpoiesis not adequately suppressed


Survival of transfused RBC in vivo

Donor (unit) FactorsAge of RBC unitsDonor geneticsG6PD

Recipient FactorsAge of patientAlloimmunizationSpleen statusComplement activation

Donor/Recipient InteractionAntigen Mismatches Antibody‐mediated

destruction (DHTR) Non‐humoral

immunologic clearance?


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


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, Fya, Jkb, alloAbs


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))


PreciseType™ HEA


PreciseType™ HEAISBT Blood Group Antigens Predicted by HEA

with licensed anti‐seraAntigens Predicted by HEA without licensed anti‐sera

RhCE C, c, E, e V, VS (possible r’S partial C)

Kell K, k Kpa, Kpb, Jsa, Jsb

Duffy Fya, Fyb Fy(a‐b‐) (GATA ‐67C)

Kidd Jka, Jkb

MNS M, N, S, s U

Lutheran Lub Lua

Diego Dia, Dib

Colton Coa, Cob

Dombrock Doa, Dob, Hy, Joa

LW Lwa, LWb

Sciana SC1, SC2


HEA‐LR BeadChip Leukoreduced (LR) RBC Unit: ≤ 5 x 106 WBC/unit Extract DNA from 3 – 4 unit segments Same panel of SNPs as PreciseType HEA Assay Does not assess SNPs associated with RHCE, RHDvariants


RHCE and RHD BeadChip Assays Identifies >75 RHD and >35 RHCE variants


Study Design

Hb S (g/dL)

Hb A (g/dL)

Months1 2 3 40

To 12 moPatient 1


∆HbA Estimation

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).

(HbA Post‐Txn) – (HbA Pre‐next Txn)( # days btwn Txn)∆HbA =

Hb A (g/dL)

days


Patient Characteristics 90 pediatric patients (88 African‐American, 2 Hispanic) Mean age 11.4 yrs (3.3 – 19.7 yrs) 1,134 transfusion episodes (2,320 LR RBC units)

At Entry At Exit

Category 1 Matching“Non‐Responders”

62 60

Category 2 Matching“Responders”

28 30

Category 1 2 Switching• 1 patient with new Ab of undetermined specificity (AUS)• 1 with historic anti‐e, found to have hrB‐ variant (ceS/ceS)


Patient Antibody HistoriesBlood Group Alloantibodies Frequency at Entry Frequency at Exit

RH C, E, e, Goa, f, V, VS, Cw 20 22(1 anti‐e, 1 anti‐Goa)

KELL K, Kpa, Jsa 9 12(3 anti‐Jsa)

Duffy Fya, Fy3 2 2

Kidd Jkb 1 1

MNS M, S 4 4

Lutheran Lua 3 3

Knops Knops 1 1

Lewis Leb 1 1

Other AUS, Wra 0 2(1 anti‐Wra, 1 AUS)

TOTALS 41 48


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 antigen‐positive units


RHCE Patient GenotypesRHCE

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)

263112

______33 (36.7%)

Potential for mismatches when transfused with antigen‐positive units


RHD Patient GenotypesRHD

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

4113____9 (10.0%)

Potential for mismatches when transfused with RHD+ units

622 TransfusionsWith reliable ΔHbA Estimates• 390 Category 1• 232 Category 1

* Due to inadequate DNA concentration

285 units without HEA• 89 HEA‐LR failure *• 196 not tested

2320 RBC units(1135 transfusions)

• 1470 Category 1• 850 Category 2

2035 units with HEA• 1293 Category 1• 742 Category 2

1892 units (932 transfusions)

143 units in a transfusion episode with ≥1 missing HEA

Transfusions evaluated for antigen

mismatches

Transfusionsevaluated for HbA

clearance


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%)

*

*

**


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?


Antigen mismatches per transfusion

0

50

100

150

200

250

0 1 2 3 4 5 6 7 8 9

Freq

uency

Antigen Mismatches per Transfusion

24.4%

20.6%

12.4%

3.1%

15.2%

13.2%

6.0%

3.4%

0.01% 0.006%


Antigen Mismatches

Fewer mismatches in Category 1 than would be expectedMay be due to selection of ethnically‐similar donors and/or donors who are phenotypically‐similar for C, E, K.

Category 1 Transfusions

Category 2Transfusions

Minimum # antigens matched

3(C/c, E/e, K)

≥5(C/c, E/e, K, Fya, Jkb, alloAbs)

Mean # of antigenMismatches

3.50 2.80



932 transfusions (627 cat 1, 305 cat 2) = 1890 units Excluding potential RHCE and RHD mismatches, due to lack of unit genotypes



932 transfusions (627 cat 1, 305 cat 2) = 1890 units Excluding potential RHCE and RHD mismatches, due to lack of unit genotypes

Matched for Cat 2

*

*

*


Alloimmunization Events 7 new alloantibodies in 5 patientsSubject ID

Category Prior Alloantibodies

CTT Duration (years)

NEW ANTIBODY

S‐1218 2 E 6.9 Jsa

E‐0108 2 S, Jkb, WAA 1.8 Go(a)E‐0117 2 AUS, Kpa, C 8 Jsa

E‐0126 2 K, E, S, Lua, WAA, AUS

10 JsaWra

H‐0101 1 none 6.1 AUS

Incidence of Alloimmunization0.706/100 units (Category 2)0.068/100 units (Category 1) p=0.02


Alloimmunization Events 7 new alloantibodies in 5 patientsSubject ID

Category Prior Alloantibodies

CTT Duration (years)

NEW ANTIBODY

S‐1218 2 E 6.9 Jsa

E‐0108 2 S, Jkb, WAA 1.8 Go(a)E‐0117 2 AUS, Kpa, C 8 Jsa

E‐0126 2 K, E, S, Lua, WAA, AUS

10 JsaWra

H‐0101 1 none 6.1 AUS

Incidence of Alloimmunization0.706/100 units (Category 2)0.068/100 units (Category 1) p=0.02

Demonstrates increased frequencyof alloimmunization in SCA patients with prior alloimmunization


Jsa Antigen Kell blood group Antigen Frequency African‐American: ~20% Caucasian <0.01% ON SCA‐CTT Study: Patients 18% Units 9% Mismatch: 13.3%

Anti‐sera for Jsa is not readily available

0.00

0.10

0.20

0.30

Patients Units Mismatch

Freq

uency

Jsa Antigen

Jsa exposures

N=49 Category 1

N=25 Category 2

3 with anti‐Jsa

22 Jsa(‐) patients

1168 units transfused 101/1014 Jsa(+) 9.96 exposures/100 units

3 new anti‐Jsa20.4 exposures/ 100 units

19 no anti‐Jsa8.33 exposures/ 100 units

636 units transfused prior to anti‐Jsa 52/546 Jsa(+) 9.52 exposures/100 units

74 (82%) Jsa(‐) patients

p=0.87

p=0.02


Jsa Alloimmunization Associated with significantly higher frequency of Jsaexposure (20.4/100 units) vs. patients without Jsaalloimmunization (8.3/100 units)

Illustrates that pattern of alloimmunization is shiftingrather 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


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)


∆HbA variation

0

0.05

0.1

0.15

0.2

0.25

1 101 201 301 401 501 601 701

ΔHbA

per tran

sfusion (g/dL/da

y)

Individual Transfusion Episodes

Mean ΔHbA = 0.084 g/dL


ΔHbA variation

0.0821 g/dL/day= 2.30 g/dL/28 days

0.0894 g/dL/day= 2.50 g/dL/28 days

p=0.0027


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 pastAlloimmunization(or autoantibodies)

Receives Category 2Transfusion matching

Patient is Immunologic“Responder”

to RBC antigens

Donor RBC (HbA‐RBC) are cleared more rapidly(possibly via non‐humoral immune mechanism)


Category 1 vs. 2 PatientsCategory 1(Non‐responders)

Category 2 (Responders)

p

Age (years), mean (SD) 10.7 (4.0) 12.5 (3.6) 0.052

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


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


HbA Clearance (ΔHbA) Category 2 transfusions (n=234): ΔHbA vs. Blood Group mismatches

pRH 0.9755KELL 0.4008KIDD 0.0273DUFFY n/aMNS 0.1385LUTH 0.2919DIEGO n/aDOMBROCK 0.1793COLTON 0.5521LW 0.4636SCIANA n/a

Note:Category 2 = matched for Jkb(not routinely for Jka)

* In few instances, Jkamatching could not be honored, due to limited blood availability when matching for rare antigens (e.g. e‐variants)


∆HbA and Kidd mismatches

Category 1 transfusions


KIDD mismatches No KIDD mismatch p

ΔHbA g/dL/day,Median

0.0811 0.0832 0.36


ΔHbA g/dL/day,Median (95% CI)

0.0986(0.093, 0.114)

0.0874(0.083, 0.092)

0.008


∆HbA and Kidd mismatches




ΔHbA g/dL/day,Median

0.0811 0.0832 0.36


ΔHbA g/dL/day,Median (95% CI)

0.0986(0.093, 0.114)

0.0874(0.083, 0.092)

0.008

While Kidd mismatches were associated with higher ∆HbA, this has not yet been validated in repeat studies at CHOA/CNMC or elsewhere


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


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)


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 African‐origin donors (Jsa, V, VS, etc) in at‐risk SCA patients?This approach could be made feasible by HEA typing of patients & donors


Clinical Applications Supports further investigation of factors that influence clearance of transfused RBC after transfusion, in order to optimize transfusionalsupport for patients with SCA


Questions?



questions


Continuing Education

• PACE, Florida and California DHS• 1.0 Contact Hours• Each attendee must register to receive CE at:

https://www.surveymonkey.com/r/12July2017Webinar

• Registration deadline is July 28, 2017

• Certificates will be sent via email only to those who have registered by August 11, 2017


Questions?



questions

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