Clinical Benefits of Red Blood Cell Genotyping...14 RHCE*ce alleles 1 RHCE*Ce allele From the 226...
Transcript of Clinical Benefits of Red Blood Cell Genotyping...14 RHCE*ce alleles 1 RHCE*Ce allele From the 226...
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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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
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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
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Application of RBC Molecular Testing in Clinical Practice
6 Hillyer CD. Transfus Med Rev 2008: 22, 117-132
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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 +
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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
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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
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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
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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
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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
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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
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Outcomes with Prospective Antigen Matching Protocols
18 LaSallle-Williams M. Transfusion 2011, 51: 1732-1739
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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
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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
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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
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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
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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 + +
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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
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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
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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
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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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