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

Transcription

1 Clinical Benefits of Red Blood Cell Genotyping Perspective from the Transfusion Service 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 1 SEABB Meeting March 20, 2014

2 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 20 th 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) 2

3 Molecular Basis of Blood Group Antigens 3 Denomme GA. Transfus Apher Sci 2011: 44, 53-63

4 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 4

5 High Throughput Red Cell Genotyping Methods 5 Veldhuisen B. Vox Sang 2009, 97:

6 Application of RBC Molecular Testing in Clinical Practice 6 Hillyer CD. Transfus Med Rev 2008: 22,

7 Emory Center for Transfusion and Cellular Therapies and Affiliates Children s Healthcare of Atlanta Emory Hospitals 7 Grady / Hughes Spalding

8 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 8

9 Phenotype Case 1: Transfusion History C E c e K k Fy a Fy b Jk a Jk b Le a Le b P 1 M N S s 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) 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

10 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 -Fy a - Jk b - and HbS negative 6/2012: new alloantibody identified 10

11 Case 1: Molecular Results Patient was confirmed to be a partial c, partial e, and hr B negative Probable RH Genotype R 0 cc variant / r variant specifically Dce733G / ce48c, 733G (RHCE* ) Predicted Phenotype (based on HEA, RHD, and RHCE genotyping) D+C-E-, partial c+, partial e+, V+, VS+, and hr B - New unit requirements: C- E- hr B - Fy a - Jk b - and HbS - 11 Reid ME. The Blood Group Antigen FactsBook 3 rd ed. 2012

12 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 12 D C E c e K k Fy a Fy b Jk a Jk b Le a Le b P 1 M N S s Blood Bank Serology : anti-e Referred for molecular testing BioArray HEA BeadChip RHD and RHCE genotyping

13 Case 2: Molecular Results Patient was confirmed to be a partial D, altered C, partial c, partial e and hr B 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+ hr B - Patient is at risk for anti-d, -C, -e, -f(ce) and - hr B 13 Flegel WA. Transfus Apher Sci 2011, 44: 81-91

14 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 14

15 Difference in Minor Antigen Prevalence between Racially Different Pairs Antigen % in white donors % in black recipients D C E c e K 9 2 Fy a Fy b Jk a Jk b S s Vichinsky EP. N Engl J Med 1990, 322:

16 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 Standard Arm 3%/unit Transfusion Arm 0.5%/unit 16 Vichinsky EP. Transfusion 2001, 41:

17 Recommendations for Prospective Antigen Matching NIH Guidelines were last updated in 2002 and endorsed the recommendation from the STOP trial 17

18 Outcomes with Prospective Antigen Matching Protocols 18 LaSallle-Williams M. Transfusion 2011, 51:

19 Additional Antigen Difference Complexity Rh variant antigens account for the second level of antigenic complexity between donor and patient RBCs Antigen % in white donors % in black recipients Partial D among D+ 1 7 Partial C among C Partial e among e+ 0 2 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 19

20 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 ( ) 58% alloimmunized 64.4% of all antibodies had specificity for common Rh antigens 20 Chou ST. Blood 2013, 122,

21 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,

22 RH Genetic Diversity in Patients 13 different RHD alleles 14 RHCE*ce alleles 1 RHCE*Ce allele with Sickle Cell Disease 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,

23 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 D C E c e K k Fy a Fy b Jk a Jk b Le a Le b P 1 M N S s Blood Bank Serology : anti-d, non-specific reactivity Referred for antibody identification and molecular testing BioArray HEA BeadChip RHD and RHCE genotyping

24 Case 3: Serology and Molecular Results Monocyte Monolayer Assay Results Cell % (0-3%) Cell % (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+) 24 Patient is at risk for anti-d, -e, -f(ce) Reid ME. The Blood Group Antigen FactsBook 3 rd ed. 2012

25 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 25

26 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

27 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 27

28 Clinical Significance of Weak D Types 28 Hillyer CD. Transfus Med Rev 2008, 22:

29 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 29

30 Questions? 30