Red Cell Transfusions in the Genomics Era

Seminars in Hematology 56 (2019) 236–240

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Red Cell Transfusions in the Genomics Era ✩ Jamal H. Carter a, Willy A. Flegel b,∗ a b

Division of Clinical Pathology/Laboratory Medicine, Department of Pathology, Montefiore Medical Center, Bronx, NY Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, Bethesda, MD

a r t i c l e

i n f o

Keywords: Red cell genotyping Molecular immunohematology Hematologic disease Transfusion Blood group Red cells

a b s t r a c t Red cell genotyping has become widely available and now contributes to support transfusion of patients with hematologic diseases. This technology has facilitated the immunohematologic approach to antibody prevention, detection and identification. Donors, particularly rare donors, are most efficiently screened and identified by red cell genotyping. In transfused patients with challenging serologic reactivity, antibodies are more reliably identified when molecular typing information is available. Red cell genotyping of both donors and patients augments the selection of blood components. This technology, serving at the core of a real-time database inventory, is resulting in blood supply efficiencies. However, there is limited published evidence on the extent to which red cell genotyping has translated into improved clinical outcomes. Red cell alloimmunized patients may benefit the most in enhanced safety. For patients with antibodies to high-prevalence antigens, other than Rh, blood centers realized supply-chain efficiencies in the past decade. Prospective clinical trials and cost-effectiveness studies would contribute to further clarifying the optimal role of molecular testing in providing transfusion support for patients with hematologic diseases. Published by Elsevier Inc.

Introduction Red cell genotyping, applied in transfusion medicine, has increased the availability and the degree of compatibility for red cell products. Its wider application has the potential to enhance care and safety, particularly for vulnerable patients with hematologic diseases. Currently, the technology is largely confined to immunohematology reference laboratories (IRLs). In this capacity, it is deployed in the profiling of a recipient’s human erythrocyte antigens (HEA), generally in the evaluation of recipient antibodies when serologic testing is challenging, or in predicting acceptable donor HEA for minor antigen matching in populations at higher risk of sensitization. Widespread adoption of these technologies, combined with large-scale donor testing regime to predict red cell component HEA, may allow the routine and timely provisioning of better matched products. The immediate impact of this development in donor testing will be in blood supply-chain

✩ Guest editors: Sandhya R. Panch, Bipin N. Savani and David F. Stroncek “Red Cell Transfusions in the Genomics Era.” ∗ Corresponding author. Willy A. Flegel, MD. Department of Transfusion Medicine, NIH Clinical Center, National Institutes of Health, 10 Center Drive-MSC 1184, Building 10, Room 1C711, Bethesda MD 20892-1184. Tel.: (301) 594-7401; fax: (301) 4969990. E-mail address: [email protected] (W.A. Flegel).

https://doi.org/10.1053/j.seminhematol.2019.11.001 0037-1963/Published by Elsevier Inc.

efficiencies, which may result in improved downstream patient care and patient safety outcomes. For example, in chronically transfused patients, the results could be less transfusion delays and a reduced risk of alloimmunization and delayed hemolytic transfusion reactions (DHTRs). Red cell sensitization in hematologic disease High rates of red alloimmunization often complicate the transfusion support for several transfusion-dependent hematologic diseases, narrowing the range of compatible units [1,2]. At the high-end of the alloimmunization prevalence spectrum are frequencies nearing, or exceeding, half of a given disease-specific patient base for a reporting institution for several conditions [1]. These include certain hemoglobinopathies, such as sickle cell disease (SCD) and β -thalassemia major (TM), and myeloid disorders, such as myelodysplastic syndromes (MDS) [1,3]. The HEA specificity of the most common red cell antibodies formed in these conditions target antigens in the RhCE and Kell blood groups, suggesting that that C, E, K prophylactic matching between recipients and donors may be able to reduce alloimmunization rates [4-6] (pp28-41). Unless sufficient interest and resources become available, this hypothesis may not be rigorously tested in multicenter randomized or pragmatic comparative effectiveness trials. Observational data of various transfusion support strategies

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Table 1 Key studies of matching strategies and immunization in patients with SCD. Study

Cohort period

Pts, N

Units, n

Matching Strategy

Immunizeda , Ni (% of N)

Ab

Ratec

Ambruso et al. (1987) [7]

1974-1984

85

NR

ABO, D

28 (33%)

66

NA

Cox et al. (1988) [8] Rosse et al. (1990) [9]

1980-1985 1979-1984

73 1814

NR NR

ABO, D ABO, D

22 (30%) 338 (19%)

29 765

NA NA

Vichinsky et al. (1990) [10] Aygun et al. (2002) [11] Castro et al. (2002) [12] Sakhalkar et al. (2005) [13] Sakhalkar et al. (2005) [13] Vinchinsky et al. (2001) [14] O’Suoji et al. (2013) [15]

1978-1985 1989-1999 1976-1995 1989-2004 1997-2004 1995-1997 2002-2011

107 140 102 387 113 61 180

1711 3239 8939 14263 2354 1830 NR

ABO, ABO, ABO, ABO, ABO, ABO, ABO,

32 (30%) 52 (37%) 102 (29%) 121 (31%) 6 (5%) 9 (15%)b 26 (14%)

68 NR 342 240 6 12b 48

4 NA 3.8 1.7 0.26 0.66b NA

Chou et al. (2013) [16] DeBaun et al. (2014) [17] Tahhan et al. (1994) [18]

1997-2012 2004-2013 1980-1993

182 90 40

44482 3236 608

E, Ke E, K E, K, Fya,

80 (44%) 4 (4%) 0

146 9 0

0.33 0.28 0

Lasalle-Williams et al. (2011) [19]

1993-2006

99

6946

ABO, D, C, ABO, D, C, ABO, D, C, Fyb, S ABO, D, C, Jka, Jkb

E (50%), K (39%), C (36%), Fya (18%), Lea (18%), S (14%), Leb (14%), Jkb (11%), M (11%) E (68%), C (31%), Leb (14%) E (42%), C (30%), K (28%), Lea (25%), Fya (18%), D (13%), Leb(12%), Jkb (11%) K (26%), E (24%), C (16%), Jkb (10%) NRPIS; CEK predominant Ab NRPIS; CEK predominant Ab NRPIS; CEK predominant Ab NRPIS; Non-CEK Ab E (2), K (2), Lea (2), e (2)b C (19%), E (15%), Kidd (13%), K (10%), S (10%) NRPIS; DCEe predominant Ab C (2), V (2), e, Fya, Jkb, S, Wra NA

E, K, Fya,

7 (7%)

16b

0.23b

e (4)b , D (4)b , M (2)

D D D D D, C, E, K D, C, E, K D, C, E, K

Ab specificities (% of Ni )d

Ab = antibodies; CEK = RhCE and Kell; NA = not available/applicable; NR = not reported; NRPIS = not reported per individual specificity; Pts = patients; SCD = sickle cell disease. a Total number of pts with RBC alloantibodies + autoantibodies with Rh specificity when indicated(b ). b Count includes warm autoantibodies with a defined Rh specificity (eg, autoanti-e) but excludes warm poly- or panspecific, similar to definitions used by Chou et al., 2013. c Calculated rate at which a new specificity is reported per 100 units. Calculated by dividing the sum of specificities by the sum of red cell transfusions for the cohort undergoing the matching protocol of interest. d Antibody specificities occurring in over 10% of the Ni . Percent of Ni in parentheses, unless Total Ni < 20, then absolute numbers of each specificity is given for specificities occurring more than once. e Exclusively from donors who self-identified as African American.

in SCD show that prophylactic matching beyond ABO and RhD is associated with reduced alloimmunization rates (Table 1) [719]. Because chronic red cell transfusion therapy is frequently fundamental to the management of SCD and TM, on the basis of these data, expert consensus guidelines currently recommend prophylactic matching for C, E, K in both SCD and TM [4,5]. Red cell genotyping Technology There have been 2 in-vitro diagnostic assays (IVDs) approved by the United States Food and Drug Administration (FDA) as tests of record (ie, do not require confirmation via serologic phenotyping). Both IVDs, PreciseType (approved May 23, 2014) and ID CORE XT (approved October 11, 2018), predict HEA by interrogating DNA polymorphisms of interest using allele-specific oligonucleotide probes. Specifically, multiplex polymerase chain reaction is used to amplify specific regions of extracted genomic DNA, followed by: hybridization to probes on the surface of color-coded beads, elongation with fluorescently labeled dNTPs, then genotype interpretation and phenotype prediction by proprietary image analysis software. These tests predict HEA distributed across over 9 different blood group systems regarded as clinically significant (Table 2) [20,21]. Clinical utility in patient diagnostics The primary benefit of these platforms in current clinical use is the ability to help in immunohematologic circumstances in which traditional serologic hemagglutination methods are difficult or impossible to perform. For example, it is often challenging to resolve red cell antibody conundrums after recent transfusion [22]. By streamlining multiantigen phenotyping, molecular testing is especially useful when serologic methods are prohibitively labor intensive, not readily available due to rare reagents, or cost-inefficient compared to high-throughput molecular testing. The published ob-

Table 2 Polymorphisms interrogated by the FDA-approved IVDs. Blood group system Polymorphism

ID CORE XT [21] PreciseType [20]

RHCE:c.122A>G RHCE:c.307T>C RHCE:c.335+3039ins109 Rh RHCE:c.676G>C RHCE:c.712A>G RHCE:c.733C>G RHCE:c.1006G>T RHD-CE-D hybrid KEL:c.578T>C Kell KEL:c.841T>C KEL:c.1790C>T SLC14A1:c.342-1G>A Kidd SLC14A1:c.838G>A SLC14A1:c.871T>C FY:c.1-67T>C Duffy FY:c.125G>A FY:c.265C>T GYPA:c.[59C>T] GYPB:c.143T>C MNS GYPB:c.230C>T GYPB:c.270+5G>T GYP.hybrid Diego DI:c.2561T>C DO:c.323G>T Dombrock DO:c.350C>T DO:c.793A>G Colton CO:c.134C>T Cartwright YT:c.1057C>A Lutheran LU:c.230A>G Landsteiner-Wiener ICAM4:c.308A>G Scianna ERMAP:c.169G>A

X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

X X X X X X X X X X X X X X X X X X X X X X X X

IVDs = in-vitro diagnostic assays; FDA = United States Food and Drug Administration.

servational experience to date, demonstrating the utility of red cell genotyping, has formed the basis for the Local Coverage Determination (LCD) by select Medicare Administrative Contractors (MACs) for one of the FDA-approved tests (Table 3) [23,24], enabling a more direct path for reimbursement by the Centers for Medicare

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Table 3 Qualifying patient categories for MAC coverage with specific IVDs [23,24]. Clinical indication

Examples provided

Allosensitization prophylaxis in chronic transfusion dependent conditions Pan-hemagglutination, or non-specific or confounded serologic reactivity Suspected antibody against an antigen with rare antisera Serologic typing discrepancies

Sickle cell anemia, Thalassemia major Warm autoantibodies, positive DAT in the setting of a recent transfusion RhD variants

MAC = medicare administrative contractors; IVDs = in-vitro diagnostic assays.

& Medicaid Services (CMS) in the MACs’ respective jurisdictions [23,24]. Antigen matching without antibodies present As suggested by the LCD indications, one proposed utility of recipient molecular HEA typing is that it may yield information, beyond serologic typing, that will be useful for prophylactic alloimmunization prevention in hematologic diseases requiring chronic transfusions [25]. While this rationale is plausible on mechanistic immunologic grounds, this potential benefit has not yet been methodically evaluated and quantified [26]. The extant observational data suggest that predicting alloimmunization risk from inferred antigenicity may not be entirely straightforward [27]. For some sensitized transfusion recipients who have rare alleles identified by molecular methods, it is unclear to what extent their molecular profile contributed to their immunization. For instance, in a report of 182 transfused SCD patients, Rh system immunization persisted despite prophylactic serologic matching for D, C, E and Kell and, more unexpectedly, occasionally occurred in patients with one or more conventional alleles on molecular profiling [16]. Nonmyeloablative allogeneic transplantation In SCD, the goal of nonmyeloablative hematopoietic progenitor cell transplantation (HPCT) is to achieve stable mixed chimerism of donor and recipient hematopoiesis in order to reverse the sickle phenotype [28]. This unusual physiologic milieu raises the concern of a greater risk of complications secondary to ABO or minor antigen mismatching: Alloimmunization, delayed engraftment, prolonged reticulocytopenia, pure red cell aplasia, and/or graft failure. In one study, immunohematologic complications were observed in 9 among 61 patients (15%) in those who underwent reduced intensity ABO-matched or ABO-minor-mismatched HPCT. Clinically, the complications ranged from nonsignificant to potentially fatal; however, the authors could not identify clinical and laboratory markers that were predictive of the complications [29]. In other work, successfully major ABO incompatible non-myeloablative HPCT succeeded in 2 patients without immunohematologic complications [30]. Current clinical trials may clarify which immunohematologic considerations are important and the role of molecular approaches [31]. Limitations Any given molecular platform’s methodology has a constraint to its depth of genotype resolution. Both PreciseType HEA and ID CORE XT report a limited number of Rh system variants (Table 2); if variant RH alleles are suspected to underlie or predispose to immunization events, a different assay with higher RH loci resolution might be desirable [32]. Currently, while there are no FDAapproved IVDs that meet stringent criteria, in practice, Rh variant detection is generally accomplished by the use of a Laboratory Developed Test (LDT) analytically validated in a laboratory certified under the Clinical Laboratory Improvement Amendment (CLIA) of 1988 as qualified to perform tests of medium or high complexity [33,34]. These considerations suggest the need for further careful

investigation to assess the utility of high-resolution molecular HEA – and, critically, Rh [25] – typing over serologic phenotyping, when prophylactic risk mitigation is a goal. Red cell genotyping of donors for transfusion support Donor database In accordance with expert-consensus guidelines in SCD and TM [4,5], many centers prophylactically match for C, E, K in the red cell transfusion support for these patient populations, using serologic phenotyping methods [33]. The greatest utility for a red cell donor registry and real-time component inventory [35], containing the molecular HEA data derived from present-day FDA-approved IVDs, would be facilitating identification of suitable antigen-negative red cell components for patients with certain rare blood needs: For example, patients who require red cell phenotypic matching beyond D, C, E and K (extended “pheno-matching”), either because they are multi-sensitized or because they have formed clinically significant antibodies targeting antigen(s) with high prevalence in the population, would most benefit [36-38]. A database, constructed for the purpose of providing rare blood to sensitized patients, could also be utilized prophylactially, to provide more extensively matched products in an effort to reduce the alloimmunization risk [39]. In the transfusion support of sensitized SCD patients, the observation of persistent Rh alloimmunization despite D, C, E matching based on serologic phenotyping of donors and recipients, unexplained by Rh variant genotypes in the recipients, suggests that this phenomenon may be explained, in part, by Rh variants in the donor [16]. This hypothesis is supported by the observation that self-identified African American blood donors show rates of variant RH alleles comparable to rates in the SCD patient population [40]. RH genotyping of red cell donors may thus facilitate providing more Rh D, C/c, and E/e compatible units in sensitized patients and reduce the volume of exposure to potentially immunizing Rh variants. However, it is unclear to what extent such a strategy, when used prophylactically, would reduce the alloimmunization rate. RH matching in hemoglobinopathies There is very little clinical information about the relative immunogenicity of, or the extent of permissiveness between, phenotypes expressed by the various RH variants at the allele, haplotype and genotype levels. Lack of such information precludes the ability to precisely predict compatibility and to accurately estimate potential efficacy of the RH molecular matching approach. Nevertheless, using virtual matching simulations, one center assessed technical feasibility by modeling a RH genotype matching strategy against current serologic D, C, E, K pheno-matching and found that the molecular route would necessitate doubling the African American donor pool [40]. In their simulations, RH genotype matching was not exclusive to RH genotype-identical matches but also included donor genotypes that were homozygous for one of the recipient’s haplotypes, and/or RH allele combinations not restricted to the haplotype arrangement found in the recipient. This matching schema, while grounded in the conventional understanding of foreign antigen immunogenicity and presumed rules of permissivity, remains to be empirically validated in the red cell antigen setting.

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Published case reports suggest that RH genotypes homozygous for one matching RH-variant-haplotype may be insufficient to avoid serologic incompatibility and/or hemolytic transfusion reactions in sensitized SCD patients with antibodies that serologically react to high-frequency Rh antigens [27,39]. This finding, while unexpected, is consistent with the similar and more commonly encountered observation that harboring one conventional RH allele does not protect SCD patients from developing antibodies that have serologic reactivity against the associated expressed conventional antigen [16]. Further study will be necessary to elucidate the precise molecular and mechanistic determinants of this immunologic phenomenon in SCD and to clarify and quantify the magnitude of clinical consequences of such incompatibility in terms of likelihood and severity of hemolytic transfusion reactions. Cost-effectiveness of red cell genotyping in SCD Comprehensive studies evaluating the cost-effectiveness of molecular HEA genotyping of either patients or donors are currently lacking. In the transfusion support of SCD, routine RH genotyping of patients and minority donors is, at present, generally regarded as cost prohibitive [40,41]. With traditional serologic methods, a Markov-based model to evaluate the cost-effectiveness of prophylactic matching for chronically transfused sickle cell patients and found C, E, K matching would cost an additional $766 million more than a history-based strategy on a national scale over 10 years, but with 2,072 fewer alloimmunization events [42]. However, such models did not capture the nonpecuniary effects of prophylactic matching, such as quality of life, survival, rate of clinically significant delayed hemolytic transfusion reactions or transfusion service delays. Cost-utility studies quantifying the impacts of matching strategies in terms of quality-adjusted life years may be ideal but are technically challenging in transfusion medicine [43]. In the future, secular trends in the cost of DNA sequencing may at some point be able to leverage widespread whole-exome and whole genome sequencing data, acquired on a given donor or patient originally for nontransfusion purposes, to minimize the direct cost burdens associated with red cell genotyping [41,44]. Use of laboratory developed tests (LDTs) Current FDA-approved platforms are limited in their Rh variant detection and would thus have poor standalone utility in this particular setting. However, in a recent non-binding guidance to industry published in December 2018, the FDA noted unapproved molecular tests may be used for HEA typing for component labeling purposes “with the approval of the responsible physician”, provided certain laboratory and testing conditions are met [45]. Rare blood inventory in the era of mass-scale donor red cell genotyping The scalability of genotyping panels coupled with a streamlined informatics pipeline and database repository have the potential for significant inventory management optimization. At one regional blood center [46], mass-scale genotyping enabled the accrual of donor typing data many times the volume of that accrued using serologic methods, in much less time [35,39]. Antigen-negative blood could be located for patients in over 90% of requests, almost 1% of which are defined as rare by the American Rare Donor Program (ARDP). Red cell genotyping enables enduring practice shifts within the blood collection service. One blood collection center stopped serologic screening on blood donors in July 2010 once a red cell genotyped inventory was established [35,46]. The most readily apparent benefit of mass-scale donor red cell genotyping may be in supply-chain efficiencies for hospital transfusion

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services, particularly those servicing patient populations with high rates of alloimmunization. Shifts in practice that occur are often considered proprietary and, thus, are not widely publicized. Further study is necessary to determine how much these system process optimizations translate into better patient outcomes and improved cost-effectiveness. Even without such data, practical financial circumstances may move more blood centers to adopt red cell genotyping approaches. Summary Red cell genotyping plays an important role in assisting in the immunohematologic investigation of patient specimens with challenging or confounding serologic reactivity, and in guiding future transfusion support. Genotyping also has the potential to facilitate the optimization of red cell component provisioning on a mass-scale by improving the efficiency by which the supply of antigen-negative units meets demand. However, the magnitude of the beneficial impact of red cell genotyping on patient outcomes, compared to traditional serology-based laboratory methods, remains to be quantified and warrants further study. Statement of disclaimer The views expressed do not necessarily represent the view of the National Institutes of Health, the Department of Health and Human Services, or the U.S. Federal Government. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Declaration of competing interest The authors declare no conflicts of interest or competing financial or personal relationships that could influence the content of this article. Acknowledgments Supported in part by the Intramural Research Program (project ID Z99 CL999999) of the NIH Clinical Center at the National Institutes of Health. References [1] Hendrickson JE, Tormey CA. Red blood cell antibodies in hematology/oncology patients. Hematology/Oncology Clinics of North America 2016;30(3):635–51. doi:10.1016/j.hoc.2016.01.006. [2] Evers D, Middelburg RA, de Haas M, et al. Red-blood-cell alloimmunisation in relation to antigens’ exposure and their immunogenicity: a cohort study. Lancet Haematol 2016;3(6):e284–92. [3] Hendrickson JE, Tormey CA. Understanding red blood cell alloimmunization triggers. Hematology 2016;2016(1):446–51. doi:10.1182/asheducation-2016.1. 446. [4] Trompeter S, Cohen A. Guidelines for the management of transfusion dependent thalassaemia (TDT). Cappellini MD, Cohen A, Porter J, Taher A, Viprakasit V, editors. 3rd Edition. Nicosia, Cyprus: Thalassaemia International Federation; 2014. [5] National Heart, Lung, and Blood Institute. evidence-based management of sickle cell disease: expert panel report (EPR), 2014. CreateSpace; 2014. [6] Lin Y, Saskin A, Wells RA, et al. Prophylactic RhCE and Kell antigen matching: impact on alloimmunization in transfusion-dependent patients with myelodysplastic syndromes. Vox Sanguinis 2017;112(1):79–86. doi:10.1111/vox.12455. [7] Ambruso DR, Githens JH, Alcorn R, et al. Experience with donors matched for minor blood group antigens in patients with sickle cell anemia who are receiving chronic transfusion therapy. Transfusion 1987;27(1):94–8. [8] Cox JV, Steane E, Cunningham G, Frenkel EP. Risk of alloimmunization and delayed hemolytic transfusion reactions in patients with sickle cell disease. Arch Intern Med 1988;148(11):2485–9. [9] Rosse WF, Gallagher D, Kinney TR, et al. Transfusion and alloimmunization in sickle cell disease. The Cooperative Study of Sickle Cell Disease. Blood 1990;76(7):1431–7. [10] Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams A, Lubin B. Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. N Engl J Med 1990;322(23):1617–21.

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