transfusion medicine in the era of blood group genomics · 1. identify the benefit of blood group...

Transfusion

Medicine in the Era

of Blood Group

Genomics

Jerry A Holmberg, PhD, MT(ASCP)SBB

Senior Director, Strategic Scientific Innovation

June 13, 2019

DG/BTS8/0519/0054

Grifols Diagnostic Solutions

Objectives

2

The participant will be able to:

1. Identify the benefit of blood group genomics (BGG) to the patient as well as to the blood

center and transfusion service.

2. Compare and contrast the differences between phenotype and genotype in transfusion

medicine, and identify the laboratory tools available today.

3. Identify areas where BGG might improve patient outcomes.

4. Discuss potential opportunities for BGG in their work environment.

BGG | Jerry Holmberg | Transfusion Medicine in the Era of Blood Group Genomics | DG/BTS8/0519/0054

1. What are blood group antigens and their functions?

Agenda

3. Cost of providing serologic workups

2. Limitations of serology

5. Blood group genotyping techniques

4. Why genotype for selected donors/patients?

What are Blood Group Antigens and

Functions?

What are blood groups antigens?

5

▪ Inherited characters present on the RBC

▪ Biochemical structure:

– Glycoprotein: amino acid sequence, oligosaccharide

– Polypeptide: amino acid chain

– Glycolipid: lipid, oligosaccharide

▪ Defined by a specific antibody:

– Alloantibody secondary to transfusion or pregnancy

– Monoclonal antibody

– Lectins


Blood group carriers within the RBC membrane

6

▪ Carbohydrates: sugars directly

absorbed on RBC membrane:

no TRUE carrier

▪ Proteins:

‒ Single pass: type 1 & type 2

‒ Multi-pass or polytopic: type 3

‒ GPI-linked: type 5


360 known RBC antigens

Recognized RBC blood groups to date

7

Blood

group

systems

Collections

Series

901

(High)

700

(Low)

36(322 antigens)

5* (14 antigens)

7 17


*Obsolete collections: 201 Gerbich; 202

Cromer; 203 Indian; 204 Auberger; 206

Gregory; 209 GLOB; 211 Wright; 212 Vel

ISBT Working Party web site accessed 2June2019

ABO, 4

MNS, 49P1PK, 3

Rh, 55

Lu, 25Kel, 36

LE, 6FY, 5

JK, 3

DI, 22YT, 5XG, 2

SC, 7

DO, 10

CO, 4

LW, 3

CH/RG, 9

H, 1

XK, 1 GE, 11

CROM, 20

KN, 9

IN, 6

OK, 3RAPH, 1

JMH, 6I, 1

GLOB, 2GIL, 1 RHAG, 3 FORS, 1

JR, 1LAN, 1 VEL, 1 CD59,

1AUG, 4

The genes encoding the 36 blood group systems are known

8

▪ 41 genes encodes the 36 blood group systems:

‒ 32 systems with single gene

‒ Rh, Xg and Ch/Rg systems have each 2 genes

‒ MNS have 3 genes

▪ 3 genes are located on allosomes: XK, XG and CD99

BGG | Jerry Holmberg | Transfusion Medicine in the Era of Blood Group Genomics |

DG/BTS8/0519/0054

Function is more than an obstacle to transfusion medicine

Blood antigens: functional molecules or obstacles

Garratty G, Telen M and Petz LD. Red Cell Antigens as Functional Molecules and Obstacles to Transfusion. Hematology 2002 445

9

Antigen (selected examples) Function

Gerbich (Ge) Membrane-cytoskeletal interaction

Chido, Rodgers (C4A, C4B) C4 component of complement

Duffy (Fy) Chemokine reception

Colton (Co) Water channel

Rh associated glycoprotein Ammonium transport

Knops-McCoy Complement receptor 1 (complement regulatory protein)

Cromer (Cr) Decay accelerating factor (complement regulatory protein)

LW Adhesion receptor

MN Chaperone of the anion channel protein (band 3)

Bg HLA class I

Diego (Di), Wright (Wr) Anion channel protein (band 3, AE1)

Kidd (Jk) Urea transporter


Blood groups are highly polymorphic

10

▪ Most blood group variations are single nucleotide polymorphism (SNP)

‒ Changes of a single base pair in the triplet codon

‒ SNPs are responsible for almost all antithetical antigenic expression

‒ Examples are C/c, Jka/Jkb, Fya/Fyb, etc.

Reid ME. Immunohematology 2008; 24(4):166-169


Other mechanisms responsible for blood group diversity

11

▪ Gene deletion: D‒

▪ Single base deletion: O

▪ SNP in transcription factor binding site: Fy-null

▪ Recombination between homologous genes: GYPB-A-B GP.Mur; RHD-CE-D D‒ VS+ V‒

NIH/National Human Genome Research Institute's


Implications of blood group antigen diversity

12

▪ Production of antibodies

▪ Destruction of homologous cells (Hemolytic Transfusion Reactions, HTRs)

‒ Acute

‒ Delayed

▪ Destruction of autologous cells

▪ Destruction of fetal cells (Hemolytic Disease of the Fetus or Newborn aka HDFN)

▪ Damage of transplanted tissues/organs


Blood collection and transfusion - annual summary for FY2017

U.S. Food and Drug Administration (FDA) fatality report

13 BGG | Jerry Holmberg | Transfusion Medicine in the Era of Blood Group Genomics | DG/BTS8/0519/0054



14

HTR (ABO) – Probable/Likely

▪ Multiple group B red blood cell units transfused

▪ Recipient initially typed as B Positive due to microscopic interpretation of the

B forward type, and absence of anti-B

▪ Retest of the original samples, the patient was determined to be O Pos,

although the back type of the patient’s plasma with B cells was markedly

weak




15

1. HTR (non-ABO): Definite/Certain ‒ Patient received emergency released-incompatible procedure (positive antibody screen)

‒ Subsequent testing confirmed anti-C, anti-Jk(a), and anti-M reactive at AHG phase

‒ Units transfused were C negative, however all were Jk(a) positive and one unit was M positive

2. HTR (non-ABO): Definite/Certain ‒ Sickle cell disease (SCD)patient with known alloantibodies including anti-C, anti-E, anti-Fy(a),

anti-Jk(b), anti-S, anti-Js(a), as well as an HTLA-like antibody and a warm autoantibody

‒ Units were negative for cognate antigens and were compatible using adsorbed plasma;

transfusion findings were consistent with a hemolytic transfusion reaction

‒ Clerical check was negative, and the transfusion reaction investigation showed no new antibodies

3. HTR (non-ABO): Definite/Certain ‒ Patient received units released under an emergency release procedure (acute intra-abdominal

hemorrhage)

‒ Positive antibody screen due to anti-e, units emergently released discovered to be e antigen

positive.

‒ Patient experienced disseminated intravascular coagulation (DIC), secondary to a non-ABO

hemolytic transfusion reaction due to anti-eBGG | Jerry Holmberg | Transfusion Medicine in the Era of Blood Group Genomics | DG/BTS8/0519/0054



16

4. HTR (non-ABO): Definite/Certain ‒ SCD patient presented with a history of anti-U and required transfusion

‒ National search was initiated, in the meantime patient required units that were emergently

released as least incompatible

‒ Patient experienced an acute hemolytic transfusion reaction due to anti-U

5. HTR (non-ABO): Possible ‒ Patient with a history of sickle beta thalassemia and anti-Fy(a)

‒ Massive transfusion units were uncrossmatched units positive for Fy(a)

‒ Patient suffered from suspected DIC and severe sickle cell crisis in addition to the non-ABO

hemolytic transfusion reaction

6. HTR (non-ABO): Possible ‒ SCD patient with no detectable alloantibodies

‒ Possibly experienced hyperhemolysis

‒ Complicated by sickle cell crisis, veno-occlusive disease and congestive heart failure


Blood collection and transfusion - summary for FY 2012‒2017



Limitations of Serology

Limitations of blood group serology (1)

19

▪ Limited availability of antisera:

‒ Commercial monoclonal antisera available for ~ 10% of antigens

‒ For ABO, Rh, Kell, Duffy, MNS only 24/144 antigens can be typed

‒ No antisera for antigens belonging to 23 blood group systems

▪ Variation in reactivity among different manufacturers

▪ Labor intensive, not adequate for extensive screening


Limitations of blood group serology (2)

20

▪ Inability to type some patients: weak antigen expression; recently transfused, warm-auto

▪ Tedious to perform crossmatch for patients with multiple antibodies, antibodies to high incidence antigens

▪ Overall limited capacity for providing extended matched blood for patients


Rates of alloimmunization in transfused recipients

Alloimmunization rate statistics

Telen, M. 2014 The Hematologist (7)

21

Disease Condition Alloimmunization Rate Alloantibodies per

Transfusion

Reference

General Population 4.4% to 10.5% Not available Summary of four studies

as reported by

Schonewille et al.

Transfusion.

2016;56;311–320

Sickle Cell Disease USA (22.33±0.13%)

versus other countries

(16.25±0.35%, p<0.000)

USA (0.45±0.003) versus

other countries

(0.20±0.005, p<0.0001)

Y. Zheng & R.W.Maitta

Transfusion Medicine,

2016, 26, 225–230.

βThalassemia Oman incidence 9.3% Not available although

anti-E (24%) and anti-K

(24%)

Al-Riyami et al.

Transfusion. 2018

doi:10.1111/trf.14508

Myelodysplastic

Syndrome or Chronic

Myelomonocytic

Leukemia

15% with incidence

rate of RBC

alloimmunization (1 per

10.5 person-years)

Not available Sanz et al. Transfusion

2013;53:710-715


Cost of Providing Serologic Workups

Centers for Medicare and Medicaid Services predict $6 trillion by 2027

U.S. health expenditures projected 2018-2027

CMS accessed 15May2019 https://www.cms.gov/research-statistics-data-and-systems/statistics-trends-and-reports/nationalhealthexpenddata/nhe-fact-sheet.html

23

▪ Projected to grow at an average rate of 5.5% per year for 2018-27 and to reach nearly $6.0 trillion by 2027

▪ Projected to grow 0.8 percentage point faster than Gross Domestic Product (GDP); as a result, the health share of GDP is expected to rise from 17.9 percent in 2017 to 19.4 percent by 2027

▪ Key economic and demographic factors fundamental to the health sector are anticipated to be the major drivers

▪ Prices for health care goods and services are projected to grow somewhat faster over 2018-27 (2.5 percent compared to 1.1 percent for 2014-17)

▪ Comparatively higher projected enrollment growth, average annual spending growth in Medicare (7.4%) is expected to exceed that of Medicaid (5.5%) and private health insurance (4.8%)

▪ Medicare enrollment impacts are the key reason the share of health care spending sponsored by federal, state, and local governments is expected to increase by 2 percentage points over the projection period, reaching 47% by 2027

▪ Insured share of the population is expected to remain stable at around 90 percent throughout 2018-27

5.5% per year increase, reaching $6 trillion by 2027


6,077 antibody positive workups in 3,608 patients at 4 majors US hospitals

Cost of serological work ups: HI-STAR study (2014)

24

▪ Data sets and cost reflects 2009-2011

▪ Most costly serologic tests:

‒ Alloabsorption: $204/test

‒ Autoabsorption: $75/test

‒ Full Ab. ID panel: $51/test

▪ Mean cost per work up: >$100 for 16/19 diagnostics:

‒ Higher: AIHA ($505/work up)

‒ Lower: NICU ($69/work up)

▪ Mean yearly cost / patient: >$180 for 15/19 diagnostics

▪ Predictors of higher cost: AIHA; Multiple transfusions; history of alloauto-antibodies

TABLE 5. Mean total cost per patient over the duration of the study

Mazonson P. et al. Transfusion 2014;54:271-277

Total cost for antibody workups

during hospital stay for AIHA

$1,490 (today adjustment $2100)


Cost of serological testing and ordering specific blood

Comparing adjusting HI-STAR with healthcare spending

Changes to Hospital Outpatient Prospective Payment and Ambulatory Surgical Center Payment Systems and Quality Reporting Programs; 21NOV2018

Mazonson et al. Transfusion 2014;54:271-277 Adjusted cost is 40% increase (5%over 8 years)

25

Test HI-STAR 2014 Adjusted Cost 2019 CMS OPPS

Antibody screen: two to four cells $7 $9.80 $50.98

Full antibody ID panel $52 $72.80 $274.22

Selected cell panel (one to four cells) $33 $46.20 $274.22 (no difference)

Selected cell panel (five to nine cells) $51 $71.40 $274.22 (no difference)

Elution $50 $70 $144.73

DAT $15 $21 $32.12

Treatment of plasma $35 $49 $274.22 (with enzymes)

Auto-adsorption $75 $105 $32.12

Allo-adsorption $204 $285.60 $32.12

Auto-control $5 $7 $32.12

Patient phenotyping (mean cost per antigen) $20 $28 $32.89

Donor blood type antigen each Not Available Not Available $274.22


Adopting blood group genotyping: is it really about the cost?

26

26

HIV

HCV

HBV

HTLV I&IIBacterial septic reaction

Palin rating scale www.riskcomm.com

TACO per

transfused patient

TACO per

transfused unit

Mistransfusion

TRALI

Allergic spectrum

Delayed HTR per transfused unit

Acute HTR per transfused unit

NAT cost $4.7-11.2

million /QALY

How much should we

pay to reduce HTR ?


Why Genotype for Selected Patients/Donors?

Waze is a GPS navigation software application owned by Google

The “WAZE” of resolving complicated transfusion workups

28BGG | Jerry Holmberg | Transfusion Medicine in the Era of Blood Group Genomics |

DG/BTS8/0519/0054

Efficiencies and cost savings in the blood bank laboratory

Reasons to perform extended blood group genotyping

▪ Roadmap of resolution to transfusion antibody complication; timely resolution of complex cases by eliminating additional steps

▪ Reduce time required to identify highly compatible units

▪ Genotype doesn’t change and can genotype a previously transfused patient; medical record

▪ Reduce testing time and reagents for complex patients:

‒ Unexpected/multiple antibodies

‒ Antigen variants: partial D, Uvar, r’s

‒ Weakly expressed antigens: weak D, Fybweak

‒ Patients undergoing monoclonal antibody therapy (anti-CD38/CD47)

‒ Recently transfused patients or DAT positive patients

▪ Obtain predicted phenotype for antigens with no antisera available (Yta, Coa, Jra)

▪ Conserve rare units

▪ Conserves RhIg for those not requiring ante or post-natal treatment


Extended period between transfusions

Benefits for frequently transfused patients (SCD)

By providing extended-matched

blood with genotyping,

transfusions were reduced from

once per week to once per month.

Da Costa DC et al. Rev Bras Hematol Hemoter. 2013;35(1):35-8.


Serological typing covers only a few antigens

Alloimmunization in repeatedly transfused patients

Proposed pathway involving RBC alloimmunization and decreased survival in patients with SCD

Nickel et al. Transfusion 2016 (56)

Transfusion

reactions

Difficulty obtaining

compatible blood

Decreased

survival

Inability to transfuse

when needed

Red blood cell

alloimmunization


DG/BTS8/0519/0054

Historical labeling of blood units using an approved test or physician sign-off

FDA guidance for industry - December 2018

32

“You should use historical antigen typing

results to label a unit only if two previous

separate donations from the donor were

tested by your blood collection

establishment and antigen typing results

were found to be concordant. The two

concordant antigen typing results may

be obtained using serological or

molecular tests or a combination

thereof.”


DG/BTS8/0519/0054

Reduced costs/time with molecular inventory

Benefits for the Laboratory/blood bank

Implementing molecular testing protocol for a readily available inventory of extended typed blood (Cedars Sinai) - 50% less expensive than purchasing antigen-negative units

1. Shafi et al, Transfusion. 2014 May;54(5):1212-9

Implementing molecular inventory

Purchasing antigen negative units

0

Annual cost comparison (x$1000)

50 100 150 200 250 300 350 400

33

Reduced time to provide antigen negative units from

more than 3 hours to less than 1 hour1


DG/BTS8/0519/0054

Blood centers are building libraries of extended genotyped units for your patients

Rare donor databases serve patients in need

34 BGG | Jerry Holmberg | Transfusion Medicine in the Era of Blood Group Genomics

Transfusion dependent

Thalassemia – chronically transfused

35

Symptoms: Fatigue, weakness, or shortness of breath; pale

appearance or a yellow color to the skin (jaundice); irritability;

deformities of the facial bones; slow growth; swollen abdomen;

dark urine

My need is Dependent on chronic transfusion therapy

My risk is Unavailability of blood when needed

Immunization to red cell antigens resulting in transfusion reactions (acute

or delayed)

Iron overload from frequent blood transfusion

What happens to me

when serology fails

Quality of life when compatible blood is unavailable and transfusion is

delayed

Hospitalization may be required

Genotyping works

because

Once genotyped, the blood bank can pre-select blood units for me based

on my genotype. Hospitalization is reduced and I don’t get over transfused.


Sickle cell disease

36

Symptoms: Fatigue and anemia; pain crisis; dactylitis

(swelling and inflammation of the hands and/or feet) and

arthritis; bacterial infections; sudden pooling of blood in the

spleen and liver congestion; lung and heart injury; leg ulcers

My need is Chronic transfusion and relief from pain when in sickle cell crisis

My risk is Death

Alloimmunization from previous transfusions causing transfusion reactions

(acute or delayed)

What happens to me

when serology fails

Treatment is delayed when compatible blood is not available due to

alloimmunization

Delayed treatment may require hospitalization with bed rest and oxygen

support

Genotyping works

because

My genotype can be tested once and part of my medical record

Blood banks can pre-select blood products to ensure compatible blood is

available

Matched genotyped blood reduces risk of future alloimmunization


https://uichildrens.org/kid-captain/2014/caitlyn-hill

Cancer with transfusion dependency

37

Symptoms: Blood in the urine; hoarseness; persistent lumps or

swollen glands; obvious change in a wart or a mole; indigestion

or difficulty swallowing; unusual vaginal bleeding or discharge;

unexpected weight loss, night sweats, or fever

My need is Transfusion dependent from cancer treatment

My risk is Death from complications of cancer and/or treatment (anti-CD38, Dara,

anti-CD47)

Alloimmunization from previous transfusions

Transfusion reactions (acute or delayed)

What happens to me

when serology fails

Compatible blood is not available when needed

Quality of life is compromised

Treatment delayed

Unknown risk of a new antibody formed if treatment like anti-CD38 used

Genotyping works

because

Once genotyped it doesn’t have to be redone

Donor blood can be matched based on genotype


Monoclonal Antibody Therapies (MAT)

38

Monoclonal Therapies with Known Interference to Immunohematology

ProceduresAntibody Drug Clinical Use Mechanism Comments

Anti-CD38 Daratumumab

MOR202

Isatuximab

Multiple Myeloma;

expanded use has

been for other

leukemia

Anti-CD38 Interferes with Indirect

Globulin Test (IAT)

Anti-CD47 Hu5F9-G4 Two types of non-

Hodgkin’s lymphoma: ▪ Diffuse large B-cell

lymphoma

▪ Follicular lymphoma

Relapse of Acute

Myeloid Leukemia

Discovered at

Stanford as a

“don’t eat me”

signal that

inhibits immune

attacks on

cancer cells

Anti-CD47 interferes with all

phases of pre-transfusion

testing, including ABO

typing. Anemia and

thrombocytopenia may

require transfusion

References available upon request


Monoclonal Antibody Therapies (MAT)

39

Monoclonal Therapies with Known Interference to Immunohematology

ProceduresAntibody Drug Clinical Use Mechanism CommentsAnti-PD1

(programmed

death) and

program death

ligand 1 therapy

Pembrolizumab

Keytruda

Nivolumab

Ventana

Melanoma

Non-Small Cell Lung

Cancer (NSCLC);Head

and Neck Squamous

Cell; Cancer (HNSCC);

Hodgkin’s Lymphoma;

and other cancers

A programmed death

receptor-1 (PD-1)-

blocking antibody ()

▪ Risk of post-allogeneic

hematopoietic stem cell

transplantation complications

▪ Lymphopenia; neutropenia,

thrombocytopenia

▪ Drug effect on T cells may

result in warm autoantibodies

Alpha 4-integrin

antagonist

Natalizumab

Tysabri

Natalizumab may be an

important addition to

the therapeutic

armamentarium for

▪ Multiple Sclerosis

▪ Crohn’s Disease

▪ ICAM 4 encodes the

Landsteiner-Wiener

(LW) blood group

antigen(s)

▪ Shares similarity

with the intercellular

adhesion molecule

(ICAM) protein

family

▪ Mild anemia to hemolytic

anemia has been reported

▪ TYSABRI was observed to

induce increases in circulating

lymphocytes, monocytes,

eosinophils, basophils, and

nucleated red blood cells

▪ May affect detection of LW

antigens

References available upon request


Blood Group Genotyping Techniques

First FDA-approved test to report genotypes as final result


DG/BTS8/0519/0054

Package inserts are available for comparison on line at www.fda.gov

FDA-cleared red cell molecular assays

1. Moulds J et al. Transfusion. 2015 Jun;55: 1418-222 2. Moulds, J. Vox Sang 42

HEA BeadChip Kit (PreciseType) ID CORE XT

Claim Intended for the molecular determination of allelic variants

that predict erythrocyte antigen phenotypes

Intended for reporting genotypes and predictive

phenotype

Predictive phenotype Yes Yes

Polymorphism results Yes but not as final interpretable result Yes

Predicted allele genotype No Yes

Hemoglobin S Yes No

RH system (Cw, hrS, hrB) No Yes

RH system ( r’S, V/VS) Use surrogate markers, 23-25% of samples positive for

these markers are not r’S1,2. Confirmatory test is required

Uses the intron 3 break point, providing and

accuracy of 100% r’S type 1 V/VS: Test position

712 instead of 941 to avoid rare polymorphism,

LW and Scianna antigens Yes No

Mia, Cartwright (Yta/Ytb) No Yes

FyB_GATA Phenotype noted as GATA silencing mutation Reported as predicted allele genotype

Limitation of target

genotype

False negative or invalid results generated by unanticipated rare mutations affecting primer or probe binding

causing allele and/or amplicon dropout. False positive or invalid results in rare cased where a sample contains

molecular events that affect blood-group antigen expression of phenotypes


DG/BTS8/0519/0054

Starting with DNA purified from patient/donor white blood cells

The ID CORE XT procedure


DG/BTS8/0519/0054

ID CORE XT results report

▪ The polymorphism result indicates which nucleotide is present; does not need to be inferred

▪ The predicted allele genotype result provides complete information of the name of the allele, complementing the information provided by the phenotype report


DG/BTS8/0519/0054

Thank you!

transfusion medicine in the era of blood group genomics · 1. identify the benefit of blood group...

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