Clinical Benefits of Red Blood Cell Genotyping...14 RHCEce alleles 1 RHCECe allele From the 226 patients genotyped, more than 1/3 of RHD and more than 1/2 RHCE allelles differed

Annie Winkler MD, MSc Assistant Professor, Emory University Department of Pathology and Laboratory Medicine

Medical Director, Grady Health System Transfusion Service Assistant Medical Director, Emory Special Coagulation Laboratory

SEABB Meeting March 20, 2014

Clinical Benefits of Red Blood Cell Genotyping Perspective from the Transfusion Service

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© Annie Winkler MD 3.16.2014 | Confidential

Introduction

Unlike other areas of the clinical laboratory, the progress of molecular pathology in transfusion medicine has been slower and more cautious

Since the discovery of the ABO blood group in the early 20th century, more than 300 authenticated blood group antigens have been placed into 33 blood group systems

The molecular bases for almost all of the blood group polymorphisms have been determined

Most antigens differ by a single nucleotide polymorphism (SNP)

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Molecular Basis of Blood Group Antigens

3 Denomme GA. Transfus Apher Sci 2011: 44, 53-63


Molecular Methods for Blood Group Antigens

Presently, there are no FDA approved molecular technologies for red cell genotyping

The methods currently available can be grouped into two categories

Low to medium throughput Low

PCR-RFLP, PCR-SSP, PCR-AS

Medium

Real-time PCR with melting curve analysis, pyrosequencing

High throughput

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High Throughput Red Cell Genotyping Methods

5 Veldhuisen B. Vox Sang 2009 , 97: 198-206


Application of RBC Molecular Testing in Clinical Practice

6 Hillyer CD. Transfus Med Rev 2008: 22, 117-132


Emory Center for Transfusion and Cellular Therapies and Affiliates

7 Grady / Hughes Spalding

Emory Hospitals

Children’s Healthcare of Atlanta

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Case 1: Clinical History 14 year old female with sickle cell disease (SCD) transferred from

Hughes Spalding to Egleston for unresponsive left arm pain due to a moderate sized joint effusion

3 admissions in the past 2 weeks for vaso-occlusive crisis and possible acute chest syndrome

The patient was also being evaluated for possible inflammatory bowel disease after findings of abdominal lymphadenopathy on CT and a positive stool guaiac were noted on a recent admission

GI was planning upper and lower endoscopies Procedures to take place at Egleston, type and screen and RBCs

ordered Hemoglobin 8.8 g/dl

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Case 1: Transfusion History Phenotype

Prior to 2/2012, the patient had negative antibody screens at

Grady/Hughes Spalding and she was receiving C-E- and HbS negative units (total of 5 RBCs from 4/2011-11/2011)

On 2/27/2012, Egleston identified and ARC confirmed an e-like antibody which was not able to be classified as auto or alloimmune Suspicion for a variant e allele

Referred for molecular testing BioArray™ HEA BeadChip™

RHCE genotyping 9

C E c e K k Fya Fyb Jka Jkb Lea Leb P1 M N S s

0 0 + + + + 0 0 + 0 0 0 + + + 0 +


Case 1: Transfusion History

With this information unbeknownst to the Grady Blood Bank, the patient was admitted to Hughes Spalding, and type and screen request for transfusion of one unit pRBCs was sent

Because of the e-like antibody, Grady issued units that were C- e -Fya- Jkb- and HbS negative

6/2012: new alloantibody identified

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Case 1: Molecular Results

Patient was confirmed to be a partial c, partial e, and hrB negative

Probable RH Genotype R0

cc variant / rvariant specifically Dce733G / ce48C, 733G (RHCE*01.20.02)

Predicted Phenotype (based on HEA, RHD, and RHCE genotyping) D+C-E-, partial c+, partial e+, V+, VS+, and hrB-

New unit requirements: C- E- hrB- Fya- Jkb- and HbS -

11 Reid ME. The Blood Group Antigen FactsBook 3rd ed. 2012


Case 2: Clinical and Transfusion History

9 year old male with SCD and history of bilateral hip avascular necrosis and acute chest syndrome required of one unit of pRBCs in preparation for removal of a left femoral plate

Transfusion History : last transfused 2011

Phenotype

Blood Bank Serology : anti-e

Referred for molecular testing BioArray™ HEA BeadChip™

RHD and RHCE genotyping 12

D C E c e K k Fya Fyb Jka Jkb Lea Leb P1 M N S s

+ + 0 + + 0 0 + + + + 0


Case 2: Molecular Results

Patient was confirmed to be a partial D, altered C, partial c, partial e and hrB negative

Probable RHD Genotype :

RHD*DIIIa-CE(4-7)-D / RHD*weak partial RHD type 4.0

Probable RHCE Genotype: RHCE*ceS / RHCE*ceS

Predicted Phenotype:

Partial D+ partial C+ E- partial c+ partial e+ V- VS+ hrB-

Patient is at risk for anti-D, -C, -e, -f(ce) and - hrB

13 Flegel WA. Transfus Apher Sci 2011, 44: 81-91


RBC Alloimmunization Pathobiology: RBC Antigen Factors

Antigenic differences between donor and recipient RBCs are requisite for the initial trigger for alloimmunization In the US, alloimmunization rates for patients with SCD range from 20-

50% in comparison to 6.1% and 2.6% in Uganda and Jamaica, respectively

Antigenic differences between donors and SCD patients have three levels of complexity 1. Prevalence of some common but highly immunogenic antigens

differs substantially between donors and transfusion recipients

2. Transfusion of Rh compatible units does not entirely prevent the risk of alloimmunization because of the prevalence of Rh variants found in persons of African descent

3. High Incidence Antigens

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Difference in Minor Antigen Prevalence between Racially Different Pairs

Antigen % in white donors % in black recipients

D 85 92

C 68 27

E 29 20

c 80 96

e 98 98

K 9 2

Fya 66 10

Fyb 83 23

Jka 77 92

Jkb 74 49

S 51 31

s 89 93

15 Vichinsky EP. N Engl J Med 1990, 322: 1617-1621


Prospective Antigen Matching Protocols Prospective phenotype matching started in single centers as early

as 1987, but were not widely adopted until the early 2000s following publication of the STOP trial Stroke Prevention Trial in Sickle Cell Anemia was a multicenter

randomized controlled trial comparing stroke risk in patients randomized to transfusion (n=63) versus standard arm (n=67) Patients were required to receive C-E-K- matched units

16 Vichinsky EP. Transfusion 2001, 41: 1086-1092

Standard Arm

3%/unit

Transfusion Arm

0.5%/unit


Recommendations for Prospective Antigen Matching

NIH “Guidelines” were last updated in 2002 and endorsed the recommendation from the STOP trial

17 http://www.nhlbi.nih.gov/health/prof/blood/sickle/sc_mngt.pdf


Outcomes with Prospective Antigen Matching Protocols

18 LaSallle-Williams M. Transfusion 2011, 51: 1732-1739


Additional Antigen Difference Complexity

Rh variant antigens account for the second level of antigenic complexity between donor and patient RBCs

Partial alleles are most often not recognized until an alloantibody has formed due to the limitations of serologic phenotyping

The third level of antigenic complexity between SCD patients and donor RBCs arises when the recipient lacks a high incidence antigen

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Antigen % in white donors % in black recipients

Partial D among D+ 1 7

Partial C among C+ 0 30

Partial e among e+ 0 2


Alloimmunization with Prospective Rh and K Phenotype Matching Chou et al recently published the results of 15 year retrospective review of

pediatric sickle cell patients at CHOP transfused using prophylactic Rh and K matching

Transfused patient characteristics Episodic 59 patients (32.4%)

Median number of RBC transfusions : 3 (1 – 15) 15% alloimmunized

Chronic 123 patients (67.6%) Median number of RBC transfusions: 230 (10 – 1460) 58% alloimmunized

64.4% of all antibodies had specificity for common Rh antigens

20 Chou ST. Blood 2013, 122, 1062-1071


Alloimmunization with Prospective Rh and K Phenotype Matching 55 (45%) chronically and 7 (12%) episodically transfused patients were Rh

alloimmunized despite prophylactic antigen matching 40% had > 1 Rh antibody 56 unexplained Rh specificities identified in 45 patients whose RBCs typed positive for the

corresponding antigen

35 unexplained Rh specificities in 33 patients whose RBCs typed negative for the antigen 40% of Rh antibodies evaluated in individuals positive for the corresponding antigen and

28% in antigen negative individuals were associated with a delayed hemolytic transfusion reaction

21 Chou ST. Blood 2013, 122, 1062-1071


RH Genetic Diversity in Patients with Sickle Cell Disease

13 different RHD alleles

14 RHCE*ce alleles

1 RHCE*Ce allele

From the 226 patients genotyped, more than 1/3 of RHD and more than 1/2 RHCE allelles differed from the conventional sequence

86% > 1 nonconventional RH allele

47.3% had >1 variant RHD and 1 variant RHCE allele

22 Chou ST. Blood 2013, 122, 1062-1071


Case 3: Patient and Transfusion History

75 year old female with a past medical history of hypertension, coronary artery disease, and chronic kidney disease was admitted for elective repair of stable abdominal aortic aneurysm (5.5. cm)

Transfusion History : no history of transfusion

Phenotype

Blood Bank Serology : anti-D, non-specific reactivity

Referred for antibody identification and molecular testing BioArray™ HEA BeadChip™

RHD and RHCE genotyping

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D C E c e K k Fya Fyb Jka Jkb Lea Leb P1 M N S s

+ 0 0 + + 0 + 0 + + 0 + +


Case 3: Serology and Molecular Results

Monocyte Monolayer Assay Results Cell 1 0-0.3% (0-3%)

Cell 2 0-0.3% (0-3%)

Auto 0.3% (0-3%)

Patient was confirmed to be a partial D and partial e Probable RHD Genotype : RHD*DIVa.2 / RHD*01N.01

Probable RHCE Genotype: RHCE*ce254G/ RHCE*48C,1025T

Predicted Phenotype:

Partial D+ C- E- c+ partial e+ Go(a+)

Patient is at risk for anti-D, -e, -f(ce)

24 Reid ME. The Blood Group Antigen FactsBook 3rd ed. 2012


Case 4: Clinical History

23 year old G2P1 with a past medical history of diabetes was seen for a routine prenatal visit and routine labs including a type and screen were sent

Type and Screen Results

Referred for molecular testing RHD genotyping

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Case 4: Molecular Results Patient was confirmed to be a weak D explaining the serologic

anti-D typing discrepancy

Probable RHD Genotype : RHD*weak D type 2 / RHD*01N.01

Predicted Phenotype: weak D+ C- E+ c+ e+

Weak D Primarily results from single

point mutations that encode amino acid changes predicted to be intracellular or in the transmembrane domain Effect the efficiency of insertion in the membrane

Over 50 different mutations have been described

26 Modified from Westhoff CM. Semin Hematol 2007, 44: 42-50


Clinical Considerations for Weak and Partial D

Of clinical concern, particularly when determining the D status of women of child-bearing age, is the distinction between a partial D and weak D phenotypes

It is important to distinguish between the two because partial D individuals may make anti-D, whereas weak D individuals are unlikely to do so

Extensive history of transfusing weak D types 1, 2, and 3 which comprise 90% of weak D phenotypes do not make anti-D and can safely receive D-positive blood and are not candidates for RhIG

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Clinical Significance of Weak D Types

28 Hillyer CD. Transfus Med Rev 2008, 22: 117-132


Conclusions

The transfusion community has moved to an exciting time where molecular technologies have emerged and are being implemented in donor centers and transfusion services

Red cell genotyping has proven to be clinically useful; however, has not been applied in large scale clinical trials

Molecular testing has entered this field, and the transfusion medicine specialist must find the ideal and cost-effective way to use this powerful tool

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Questions?

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Documents

Clinical Benefits of Red Blood Cell Genotyping...14 RHCE*ce alleles 1 RHCE*Ce allele From the 226 patients genotyped, more than 1/3 of RHD and more than 1/2 RHCE allelles differed

Clinical Benefits of Red Blood Cell Genotyping...14 RHCEce alleles 1 RHCECe allele From the 226 patients genotyped, more than 1/3 of RHD and more than 1/2 RHCE allelles differed