Abstract
Background.
Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part due to a high frequency of variant RH alleles in patients with SCD and Black blood donors.
Study Design and Methods.
We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the United States. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays.
Results.
Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5–41% (p=0.0035), and 5–33% (p=0.0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p=0.0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens.
Discussion.
These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.
Introduction
Multicenter studies have identified a 2–5% prevalence of red cell alloimmunization in the general population, while in patients with SCD it ranges from 5–75%.1,2 Despite serological Rh antigen matching, alloimmunization persists due to the high frequency of RH variants among both patients with SCD and Black donors.3–6 Over 25% of patients with SCD have RH variant alleles resulting in partial D, C, and/or e antigens that lack common epitopes and can lead to recognition of a conventional protein as foreign.7,8 Individuals with exclusive partial D expression are more likely to form anti-D with fewer D+ unit exposures, and these antibodies persist longer than in patients with conventional RhD, whose antibodies are likely due to exposure to donor RBCs with variant RH.5 While most RH variants found in Blacks result in partial antigen expression, RHD*DAU0 (RHD*10.00) encodes one amino acid change (p.Thr379Met) that does not result in loss of a RhD epitope and has a frequency of 16% in Blacks.9 Similarly, RHCE*ce(48C) (RHCE*01.01) encodes one amino acid change (p.Trp16Cys), and although reported to lack reactivity with some monoclonal anti-e,10 it has not been associated with production of clinical significant allo-anti-e. For a RH genotype matching strategy, considering these alleles as equivalent to their corresponding conventional alleles increases the number of suitable Black donors for RH matching.11
Rh variant antigens can be difficult to define serologically, therefore, some advocate for RH genotyping to identify those with partial antigens for whom donor selection can be modified.12 In addition, unexpected Rh antibodies occur in patients with conventional RH alleles or in those who received antigen-negative units,4,11 implicating donor red cells expressing variant Rh as the cause of alloimmunization.6 Thus, identifying RH variants among both patients and donors is important, and RH genotype-matched red cells may be necessary for optimal prevention of alloimmunization.6 In the current study, we compare RH genotypic variability and Rh alloimmunization among 342 chronically transfused patients with SCD from geographically distinct academic centers across the US who cumulatively received >90,000 red cell exposures from regional donors.
Patients and Methods
Under institutional review board-approved protocols, blood samples or DNA were obtained from patients with SCD at 5 sites across the US: Children’s Hospital of Philadelphia (CHOP), n=173 patients, in Philadelphia, PA; Children’s Healthcare of Atlanta (CHOA), n=73 patients, in Atlanta, GA; St. Jude Children’s Research Hospital (SJCRH), n=60 patients, in Memphis, TN; UH Rainbow Babies and Children’s Hospital (UHRBCH), n=20 patients, in Cleveland, OH; and University of San Francisco (UCSF) Benioff Children’s Hospital Oakland, n=16 patients, in Oakland, CA. Each site is located in urban areas with populations varying from 430,000 to 1.56 million and percentage of Black individuals ranging from 22% to 65%.13 Inclusion criteria were a diagnosis of SCD and receipt of chronic red cell transfusion therapy, defined as regularly scheduled transfusions every 3 to 6 weeks. All sites prophylactically matched for ABO, Rh (D, C, E) and K by serologic type. At CHOP, CHOA, UCSF and UHRBCH, patients received C, E and/or K antigen negative units if they lacked the antigen. At SJCRH, patients mainly received C, E, and K antigen negative units regardless of antigen status; if unavailable, patients were matched based on their antigen status. At CHOA and UCSF, alloimmunized patients received extended matching for Duffy (Fy) and Kidd (Jk), or Fy, Jk and MNS antigens, respectively. At all sites, patients received units lacking the antigen(s) to which they had formed antibodies, if possible. Black donor units were preferentially selected for patients at CHOP. All institutions provided leukoreduced and hemoglobin S-negative units. CHOP, SJCRH and UHRBCH universally irradiated red cell units, while CHOA and UCSF only provided irradiated units if patients were proceeding to transplant or gene therapy.
Retrospective review of clinical records from birth of each patient through July 2018 was performed to document the patient’s red cell antigen phenotype obtained by serology or genotype, the number of units exposed, and the alloimmunization history. Antibody identification was performed according to institutional practice and included tube, solid-phase, and gel-based methods. Antibodies considered clinically insignificant included: anti-Bga, anti-I, anti-M non-reactive at 37C, anti-Sda and high-titer low-avidity antibodies. Warm and cold autoantibodies and antibodies of undetermined significance were excluded from the alloantibody totals. Data coordination and statistical analysis were performed at CHOP.
RH genotyping
Blood samples or DNA from all sites were sent to the New York Blood Center (NYBC), where DNA was isolated by routine methods (QIAamp; QIAGEN, Valencia, CA). Genotyping was performed with RHD and RHCE BeadChip arrays (Immucor, Warren, NJ) and polymerase chain reaction (PCR) assays as performed previously.4,11 See supplemental methods for additional details.
We defined “altered” Rh antigen expression on the red cells of patients with one or two RHD*DAU0 (RHD*10.00) or RHCE*ce(48C) (RHCE*01.01) alleles and no conventional allele; this terminology was used to distinguish them from individuals who express only partial Rh and lack Rh epitopes.
Statistical Analysis
The Kruskal-Wallis rank sum test was used to compare median age of individuals and median number of units transfused to each patient per site (Table 1). Local polynomial regression and Spearman’s correlation were used to adjust for the impact of age on number of units transfused. Contingency table analyses were used to compare frequencies among categorical variables. Pearson’s chi-squared test was primarily utilized throughout the study to compare three or more categorical variables. In circumstances with only two categorical variables, Fisher’s exact test was performed.
Table 1. Demographics and Alloimmunization Rates.
Patient median age and median number of units transfused to patients were evaluated using the Kruskal-Wallis rank sum test. Total alloantibodies across the five sites are listed. Prevalence and incidence rate of overall and Rh alloimmunization were calculated using Pearson’s chi-squared test.
| CHOP n=173 | CHOA n=73 | SJCRH n=60 | UHRBCH n=20 | UCSF n=16 | p value | |
|---|---|---|---|---|---|---|
| Median Age in Years (Range) | 11 (0–39) | 12 (3–21) | 14 (4–21) | 12 (1–16) | 22 (11–52) | <0.0001 |
| Total # Units Exposed to All Patients | 62032 | 15054 | 5348 | 2291 | 5327 | |
| Median # Units Exposed Per Patient | 114 | 183 | 75 | 113.5 | 286.5 | <0.0001 |
| Total alloantibodies | 186 | 44 | 10 | 1 | 5 | |
| Total # of Patients Alloimmunized (%) | 71 (41.0) | 21 (28.8) | 8 (13.3) | 1 (5.0) | 3 (18.8) | 0.0035 |
| Overall Alloimmunization Incidence Rate (Antibodies per 100 units transfused) |
0.300 | 0.292 | 0.187 | 0.044 | 0.094 | 0.0075 |
| Overall Alloimmunization Incidence Rate Subgroup Analysis (Antibodies per 100 units transfused) |
0.300 | 0.292 | 0.183 | - | - | 0.3412 |
| Total # of Patients Rh Alloimmunized (%) | 57 (33.0) | 14 (19.2) | 5 (8.3) | 1 (5.0) | 2 (12.5) | 0.0002 |
| Rh Alloimmunization Incidence Rate (Antibodies per 100 units transfused) |
0.166 | 0.133 | 0.112 | 0.044 | 0.038 | 0.0846 |
Results
Patient Characteristics
The transfusion history was obtained from 342 individuals with SCD who underwent chronic, regular red cell transfusion: 173 patients at CHOP, 73 at CHOA, 60 at SJCRH, 20 at UHRBCH, and 16 at UCSF (Table 1). The median age of individuals at each site was 11, 12, 14, 12, and 22 years old, respectively (p<0.0001). The total number of units transfused varied considerably among the sites, ranging from 2,291 units provided to 20 patients at UHRBCH to 62,032 units transfused to 173 patients at CHOP. The median number of units transfused to each patient per site ranged from 75 to 287; individuals at UCSF had a significantly higher transfusion burden (p<0.0001), which correlated with the higher median age. After adjustment for age using local polynomial regression, there was no difference in transfusion burden among sites (R=−0.07, p=0.21).
Alloimmunization
The overall prevalence of alloimmunized patients in this study was 30.4% but varied by site: 41% at CHOP, 28.8% at CHOA, 13.3% at SJCRH, 5% at UHRBCH, and 18.8% at UCSF (Table 1) (p=0.0035). The overall alloimmunization rate also varied significantly between institutions: 0.300 antibodies per 100 units transfused at CHOP, 0.292 at CHOA, 0.187 at SJCRH, 0.044 at UHRBCH and 0.094 at UCSF (Table 1) (p=0.0075). A subgroup analysis that compared overall alloimmunization rate between the three sites enrolling the most patients, CHOP, CHOA and SJCRH, found no significant difference in antibodies per 100 units transfused (Table 1) (p=0.3412).
Overall, Rh alloimmunization prevalence was 23.1% but varied significantly: 33% of patients at CHOP, 19.2% at CHOA, 8.3% at SJCRH, 5% at UHRBCH, and 12.5% at UCSF (Table 1) (p=0.0002). The Rh alloimmunization rate was not significantly different among sites: 0.166 Rh antibodies per 100 units transfused at CHOP, 0.133 at CHOA, 0.122 at SJCRH, 0.044 at UHRBCH and 0.038 at UCSF (p=0.0846).
Among all 342 patients who received prophylactic Rh and K serological matched red cells across 5 sites, a total of 132 Rh antibodies were identified in 78 patients: 35 anti-D, 31 anti-C, 20 anti-E, 21 anti-e, 7 anti-Goa, 6 anti-V, 4 anti-VS, 2 anti-hrB, 1 anti-f, 1 anti-Rh32, 4 anti-CW (Table 2). Rh antibodies comprised 55.4% of antibodies identified at CHOP (n=103), 45.5% at CHOA (n=20), and 60% at SJCRH (n=6). The only Rh antibody identified at UHRBCH was an anti-D, and at UCSF, one anti-C and one anti-V. Overall, greater than half of all antibodies (53.7%) were directed against the Rh system (Table 2).
Table 2. Rh Antibodies.
Absolute numbers of each Rh antibody identified across the five sites. Total Rh antibodies and the percentage of Rh antibodies from all clinically significant antibodies are listed.
| Rh Antibody | CHOP n=173 | CHOA n=73 | SJCRH n=60 | UHRBCH n=20 | UCSF n=16 | All Sites n=342 |
|---|---|---|---|---|---|---|
| Anti-D | 28 | 2 | 4 | 1 | 0 | 35 |
| Anti-C | 25 | 3 | 2 | 0 | 1 | 31 |
| Anti-E | 15 | 5 | 0 | 0 | 0 | 20 |
| anti-e | 19 | 2 | 0 | 0 | 0 | 21 |
| anti-Goa | 4 | 3 | 0 | 0 | 0 | 7 |
| Anti-V | 4 | 1 | 0 | 0 | 1 | 6 |
| Anti-VS | 3 | 1 | 0 | 0 | 0 | 4 |
| Anti-hrB | 1 | 1 | 0 | 0 | 0 | 2 |
| Anti-f | 1 | 0 | 0 | 0 | 0 | 1 |
| anti-Rh32 | 1 | 0 | 0 | 0 | 0 | 1 |
| anti-CW | 2 | 2 | 0 | 0 | 0 | 4 |
| Total Rh antibodies (% total) | 103 (55.4) | 20 (45.5) | 6 (60) | 1 (100) | 2 (40) | 132 (53.7) |
RH genotypes and predicted antigen expression
RH genotyping of this chronically transfused cohort of patients demonstrated that 33% of RHD and 57% of RHCE alleles were partial or altered (Tables 3, 4). RHD and RHCE allele frequency among individual study sites was similar (p=0.7027 and p=0.2989, respectively). There was a significant difference among sites in allele frequencies of RHD*weak partial 4.0 (RHD*09.03) and RHD*DIIIa (RHD*03.01) alleles (Table 3) (p=0.0359 and p=0.0083, respectively) and in the frequency of RHCE*cE(48C) (RHCE*03.18), which was found in only one patient from CHOP (Table 4) (p=0.0029). The entire cohort reflected previously described frequencies in patients with SCD (Supplemental Table 1, p=0.9593), though there was an increase in the frequency of RHCE*ce(48C) (RHCE*01.01) alleles identified compared to prior published data (Supplemental Table 2, 0.1931 vs. 0.2412, p=0.0102).11 The allele frequency of RHD*DIIIa-CE(4–7)-D (RHD*03N.01) and RHD*weak partial 4.0 (RHD*09.03), two common alleles in this population encoding partial RHCE and RHD alleles, mirrored previous reports.3 The allele frequencies of RHCE*ce(733G) (RHCE*01.20.01), RHCE*ce(48C,733G) (RHCE*01.20.02.01), and RHCE*ce(254G) (RHCE*01.06.01) were also similar to prior reports.3 Additionally, RHD and RHCE allele frequencies in chronically transfused patients with SCD did not differ significantly from a cohort of patients who did not receive chronic transfusion therapy (Supplemental Tables 3, 4; p=0.9232, p=0.3562, respectively).
Table 3. RHD allele frequency by hospital site.
A comparison of RHD allele frequencies between five sites was performed using Pearson’s chi-squared test. Partial or altered RHD alleles make up 33% of alleles at all sites. There was no significant difference in overall RHD allele frequencies (p=0.7027).
| RHD* gene | Total # (n=684) | Frequency (all sites) | CHOP (n=346) | CHOA (n=146) | SJCRH (n=120) | UHRBCH (n=40) | UCSF (n=32) | p value |
|---|---|---|---|---|---|---|---|---|
|
RHD
(RHD*01) |
353 | 0.5160 | 0.5029 | 0.5479 | 0.4917 | 0.5500 | 0.5625 | 0.8121 |
|
DAU0
(RHD*10.00) |
124 | 0.1812 | 0.1763 | 0.2055 | 0.1583 | 0.2500 | 0.1250 | 0.5553 |
|
Deleted D
(RHD*01N.01) |
105 | 0.1535 | 0.1590 | 0.1301 | 0.1750 | 0.1500 | 0.1250 | 0.8574 |
|
inactive RHD Ψ
(RHD*08N.01) |
19 | 0.0277 | 0.0260 | 0.0205 | 0.0500 | 0.0250 | 0.0000 | 0.4867 |
| DIIIa-CE(4–7)-D (RHD*03N.01) | 16 | 0.0233 | 0.0231 | 0.0274 | 0.0333 | 0.0000 | 0.0000 | 0.6722 |
|
Weak partial 4.0
(RHD*09.03) |
15 | 0.0219 | 0.0347 | 0.0000 | 0.0083 | 0.0000 | 0.0625 | 0.0359 |
|
DAU3
(RHD*10.03) |
14 | 0.0204 | 0.0173 | 0.0205 | 0.0333 | 0.0000 | 0.0313 | 0.7025 |
|
DIIIa
(RHD*03.01) |
11 | 0.0160 | 0.0145 | 0.0068 | 0.0167 | 0.0000 | 0.0938 | 0.0083 |
|
DIVa
(RHD*04.01) |
10 | 0.0146 | 0.0173 | 0.0137 | 0.0167 | 0.0000 | 0.0000 | 0.8631 |
|
DAU5
(RHD*10.05) |
9 | 0.0131 | 0.0116 | 0.0274 | 0.0083 | 0.0000 | 0.0000 | 0.4744 |
|
DAR
(RHD*09.01) |
2 | 0.0029 | 0.0029 | 0.0000 | 0.0000 | 0.0250 | 0.0000 | 0.1091 |
|
DUC3
(RHD*01.01) |
1 | 0.0014 | 0.0000 | 0.0000 | 0.0083 | 0.0000 | 0.0000 | 0.3187 |
|
DFR
(RHD*17.01) |
1 | 0.0014 | 0.0029 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.9131 |
| New RHD*Ψ | 1 | 0.0014 | 0.0029 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.9131 |
| RHD*835A | 1 | 0.0014 | 0.0029 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.9131 |
|
DWN
(RHD*49) |
1 | 0.0014 | 0.0029 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.9131 |
|
DFV
(RHD*08.01) |
1 | 0.0014 | 0.0029 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.9131 |
Table 4. RHCE allele frequency by hospital site.
A comparison of RHCE allele frequencies between five sites was performed using Pearson’s chi-squared test. Partial or altered RHCE alleles make up 57% of alleles at all sites. There was no significant difference in overall RHCE allele frequencies (p=0.2989).
| RHCE* gene | Total # (n=684) | Frequency (all sites) | CHOP (n=346) | CHOA (n=146) | SJCRH (n=120) | UHRBCH (n=40) | UCSF (n=32) | p value |
|---|---|---|---|---|---|---|---|---|
|
ce
(RHCE*01) |
161 | 0.2353 | 0.2139 | 0.1849 | 0.2000 | 0.2250 | 0.2813 | 0.7922 |
|
ce(48C) (RHCE*01.01) |
165 | 0.2412 | 0.2659 | 0.2671 | 0.3000 | 0.2000 | 0.2500 | 0.8031 |
|
ce(733G) (RHCE*01.20.01) |
82 | 0.1198 | 0.1069 | 0.1575 | 0.1333 | 0.1250 | 0.0313 | 0.2762 |
|
Ce
(RHCE*02) |
69 | 0.1008 | 0.1040 | 0.0959 | 0.1167 | 0.0750 | 0.0625 | 0.8743 |
|
cE
(RHCE*03) |
68 | 0.0990 | 0.1098 | 0.0822 | 0.0667 | 0.1750 | 0.0938 | 0.2973 |
|
ce(48C,733G)
(RHCE*01.20.02.01) |
39 | 0.0570 | 0.0549 | 0.0479 | 0.0583 | 0.0250 | 0.1563 | 0.1424 |
|
ce(254G)
(RHCE*01.06.01) |
39 | 0.0570 | 0.0578 | 0.0616 | 0.0417 | 0.0750 | 0.0625 | 0.9321 |
|
ceS
(RHCE*01.20.03) |
25 | 0.0365 | 0.0376 | 0.0274 | 0.0500 | 0.0000 | 0.0625 | 0.5407 |
|
ceMO
(RHCE*01.07) |
12 | 0.0175 | 0.0173 | 0.0342 | 0.0000 | 0.0250 | 0.0000 | 0.2667 |
|
ceTI
(RHCE*01.02.01) |
12 | 0.0175 | 0.0202 | 0.0205 | 0.0167 | 0.0000 | 0.0000 | 0.8244 |
|
ceAR
(RHCE*01.04.01) |
2 | 0.0029 | 0.0029 | 0.0000 | 0.0000 | 0.0250 | 0.0000 | 0.1091 |
|
ceCF
(RHCE*01.20.06) |
2 | 0.0029 | 0.0029 | 0.0000 | 0.0083 | 0.0000 | 0.0000 | 0.7645 |
|
CeCW
(RHCE*02.08.01) |
2 | 0.0029 | 0.0000 | 0.0068 | 0.0000 | 0.0250 | 0.0000 | 0.0632 |
|
ceTI type 2
(RHCE*01.20.04.01) |
1 | 0.0014 | 0.0000 | 0.0068 | 0.0000 | 0.0000 | 0.0000 | 0.4495 |
|
ce(254G,733G)
(RHCE*01.06.02) |
1 | 0.0014 | 0.0000 | 0.0068 | 0.0000 | 0.0000 | 0.0000 | 0.4495 |
|
ceHAR
(RHCE*01.22.01) |
1 | 0.0014 | 0.0029 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.9131 |
|
cEIV
(RHCE*03.04) |
1 | 0.0014 | 0.0029 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.9131 |
|
cE(48C) (RHCE*03.18) |
1 | 0.0014 | 0.0000 | 0.0000 | 0.0000 | 0.0250 | 0.0000 | 0.0029 |
Among 342 patients, 21 patients exclusively expressed partial D among 282 D+ individuals (7.5%) and may be at risk for anti-D (Figure 1A).5 A total of 44 D+ patients (12.9%) had at least one RHD*DAU0 (RHD*10.00) allele without a conventional RHD (RHD*01), which has not been associated with risk for clinically significant anti-D.5 A total of 16 of 81 C+ (19.8%), 53 of 338 c+ (15.7%), 1 of 67 E+ (1.5%) and 55 of 338 (16.3%) e+ individuals expressed the corresponding partial Rh antigen only (Figure 1A). An additional 98 c+ (29.0%) and 100 e+ (29.6%) patients did not have a conventional RHCE*ce, *cE, or *Ce alleles but carried at least one RHCE*ce(48C) (RHCE*01.01) respectively which has not been associated with clinically significant alloantibody production.
Figure 1. Rh antigen expression predicted by genotype.
Distribution of Rh antigen expression and number of patients with Rh antibodies in the current study. A) Rh antigen expression as predicted by RH genotype of 342 patients across all sites, expressed as percentage of total patients. B) Number of patients with indicated Rh antibody specificity and their corresponding antigen expression predicted by RH genotype. Legend: Negative indicates that the patient had no conventional, partial or altered alleles. Partial indicates that the patient had only partial alleles, without conventional or altered alleles. DAU0 or ce48C indicates that the patient had either of those alleles, without conventional alleles. Conventional indicates the patient had one or two conventional RHD or RHCE alleles.
RH genotyping identified loss of the high prevalence Rh antigens hrB in 38 (11%) and hrS in 7 (2%) patients (Figure 1A). Of note, 2 individuals lacking hrB had formed anti-hrB but no anti-hrS were identified in those lacking the antigen (Figure 1B). V and VS are low prevalence Rh antigens but are common among Black individuals.14 In the study cohort, 116 (33.9%) and 137 (40.1%) patients expressed V and VS, respectively, (Figure 1A) and 10 anti-V or -VS were identified (Figure 1B).
Rh antigen expression was similar between all 5 hospital sites, including expression of D, C, c, E, V, VS, hrB or hrS antigens (Supplemental Figure 1). Only expression of e antigen showed significant variability (overall p=0.0068). Prevalence of conventional e antigen varied between 48–62% among sites (p=0.0085), and the frequency of patients with e antigen expressed from one or two RHCE*ce(48C) (RHCE*01.01) alleles but no conventional RHCE*ce (RHCE*01) ranged from 15–37% (p<0.001). There was no difference between prevalence of partial e antigens or patients lacking e antigen.
Examination of Rh antibodies with predicted antigen expression
We examined each Rh antibody and the patient’s predicted antigen expression to determine the associations between having conventional, partial, or altered Rh expression and prevalence of Rh antibodies (Figure 1B, Table 5).
Table 5. Prevalence of Rh antibodies by predicted Rh antigen expression.
Patients with anti-D, -C, -E and -e antibodies were evaluated using Pearson’s chi-squared test to determine associations between respective antigen expression and antibody formation.
| Antibody | Antigen Expression | Patients with at least one conventional antigen (% total) | Patients with partial antigen only (% total) | Patients with altered antigen and without conventional antigen (% total) | Patients negative for antigen expression (% total) | p value | X2, df |
|---|---|---|---|---|---|---|---|
| Anti-D | D | 10.0 | 19.0 | 9.0 | 6.3 | 0.5447 | 2.136, 3 |
| Anti-C | C | 3.1 | 0.0 | - | 11.1 | 0.0565 | 5.748, 2 |
| Anti-E | E | 7.6 | 0.0 | - | 5.5 | 0.7835 | 0.488, 2 |
| Anti-e | e | 3.1 | 18.2 | 5.4 | 0.0 | 0.0007 | 17.16, 3 |
Anti-D.
Anti-D was identified in 34 of 326 D+ and 1 of 16 D− patients. Four of the 34 immunized D+ individuals had RHD alleles encoding partial D antigen only, and four other D+ individuals had one or two RHD*DAU0 (RHD*10.00) alleles and no conventional RHD*RHD (RHD*01) allele. The remaining 26 D+ patients with anti-D had one or two conventional RHD*RHD (RHD*01) alleles. Anti-D was identified in 10.0% (26/261) of patients with at least one RHD allele encoding a conventional D antigen, compared to 19.0% (4/21) of patients encoding only a partial D antigen (Supplemental Table 5), and 9.0% (4/44) of patients encoding one or two RHD*DAU0 (RHD*10.00) alleles without conventional D (Table 5) (p=0.5447).
Anti-C.
Among 31 patients with anti-C, 2 patients were C+ with a conventional RHCE*Ce (RHCE*02) allele. The remaining 29 patients were C− and had received C-negative blood. Anti-C was formed by 11.1% (29/261) of patients who were C−, compared to 3.1% (2/63) of C+ patients (Table 5) (p=0.0565).
Anti-E.
Among 20 patients with anti-E, 5 patients were E+ and had a conventional RHCE*cE (RHCE*03) allele. The remaining 15 patients had no alleles that encoded an E antigen and had received E-negative blood. Anti-E was formed by 5.5% (15/274) of patients who were E−, compared to 7.6% (5/66) of E+ patients (Table 5) (p=0.7835).
Anti-e.
Anti-e was identified in 21 patients at CHOP and CHOA. Six patients had at least one conventional RHCE*ce (RHCE*01) or RHCE*Ce (RHCE*02) allele. Ten patients had RHCE alleles encoding only partial e antigen, while five patients had one or two RHCE*ce(48C) (RHCE*01.01) alleles but no conventional RHCE allele in trans. Anti-e was formed by 3.1% (6/191) of patients with an RHCE allele encoding at least one conventional e antigen, compared to 18.2% (10/55) of patients encoding only partial e antigen (Supplemental Table 5), and 5.4% (5/92) of patients with one or two RHCE*ce(48C) (RHCE*01.01) alleles and no conventional RHCE alleles (Table 5) (p=0.0007).
Other anti-Rh.
Six anti-V and 3 anti-VS were detected in antigen negative individuals. Interestingly, one anti-VS was reported in a patient whose RH genotype predicts VS+ status. One anti-f was identified in a patient with RHCE*ce(733G) (RHCE*01.20.01) and RHCE*ce(48C,733G) (RHCE*01.20.02.01) alleles predicted to express partial f (ce). Seven anti-Goa, 4 anti-CW, 2 anti-hrB and 1 anti-Rh32 occurred in patients whose RH genotypes confirmed they lacked those antigens (Figure 1B).
Discussion
This study aimed to investigate the prevalence of red cell alloimmunization and RH genotypes in patients with SCD receiving chronic red cell transfusions across geographically distinct sites in the US. With 342 patients exposed to >90,000 red cell units, it is, to our knowledge, the largest cohort of chronically transfused patients with SCD with comprehensive RH genotypes and alloimmunization history. We highlight variant RH frequency, variability in alloimmunization, and the association of variant antigen expression with antibody formation.
Rh alloimmunization prevalence varied from 5% to 33%; however, overall alloimmunization rates per 100 units transfused were similar between three sites (0.300, 0.292 and 0.187), but was significantly lower at two other sites (0.044 and 0.094). These findings suggest that beyond RH genetic diversity among patients, other factors contribute to the observed differences. Slightly different antigen matching strategies at each of the 5 sites may have impacted alloimmunization rates. Alloimmunized patients at CHOA and UCSF received extended matching for Fy, Jk, or Fy, Jk and MNS antigens, respectively. SJCRH primarily provided C, E and K antigen negative units regardless of antigen status. At CHOP, Black donors were specifically recruited by their supplier for patients with SCD, although other suppliers likely preferentially C, E, and K type Black donors for selection. Lastly, the antibody identification methods varied, with solid-phase, gel and tube-based methods being used.
Donor attributes likely contribute to the variation in alloimmunization rate observed. While donor race was not available due to the retrospective nature of the study, blood suppliers for each site were typically regional and the proportion of units from Black donors at each site may reflect the local population. At CHOP, units primarily came from Black donors collected in the New Jersey and Pennsylvania region. CHOA and SJCRH were supplied by donor centers in Georgia, Florida, and Alabama, or Mississippi and Tennessee, respectively. Among these three sites with higher rates of alloimmunization, the proportion of Black individuals from these geographic regions was 41 to 65%13. In contrast, USCF was supplied from donor centers in Northern California, while UHRBCH was supplied from donor centers in Cleveland, where the proportion of Black individuals in the population of these regions varied between 22 to 47%, respectively13. These differences among donor populations potentially contribute to the variation in alloimmunization rates observed. However, despite national efforts to increase minority donors, the proportion of Black, Hispanic, or Asian donors does not parallel the diversity of the United States population, and thus, the availability of Black donors at each of study sites is still unknown15.
A significant proportion of RHD and RHCE were variant in this patient cohort: 33% RHD and 57% RHCE alleles. Patients with RHCE encoding partial e antigens had higher rates of anti-e compared to those encoding conventional e (18.2% vs. 3.1%, Table 5). However, there was no significant difference in the formation of anti-D, -C, or -E among patients with predicted partial antigen expression and those with conventional or altered antigens. While anti-D formation was not statistically different among groups, there was a trend towards an association of patients with partial D antigen expression and RhD immunization, consistent with a recent report.5 Anti-D was found in a higher proportion of patients from CHOP with conventional RHD, who received units primarily from Black donors, compared to the remaining sites (15.6% vs. 0–6.7%), but the unit exposure burden for these patients was also higher. These findings confirm the alloimmunization risk for a patient with partial antigen expression, but also suggest variant Rh on donor red cells may be stimulating alloimmunization events in recipients with conventional Rh.
The substantial number of Rh antibodies in patients who either express conventional antigen or receive antigen negative red cells in a prophylactic matching strategy suggests that donors with Rh variants are contributing to alloimmunization. Black donors have similar variant RH frequencies as patients,4 hence recipients are frequently exposed to these foreign epitopes. A case series of seven patients from Brazil provided evidence that patients exposed to RBC units from donors with Rh variants were at risk of forming allo-Rh antibodies.16 We recently reported a patient who demonstrated anti-C after receiving only C-negative units; RH genotyping of donors revealed transfusion of a Black donor unit who was compound heterozygous for variant RHD and RHCE: RHD*DIIIa/weak partial 4.0 (RHD*03.01/09.03) and RHCE*ceS/ce(48C,733G) (RHCE*01.20.03/01.20.02).6 This genotype predicts a partial D+, partial e+, V+VS+, DAK+, and hrB− phenotype and is not known to express C-like epitopes. We hypothesize that the variant RH combination results in a structural conformation change that caused the sensitization and highlights the complexity of RH variants.
Prophylactic Rh and K antigen matching reduces alloimmunization rates to 0.26–0.50 antibodies per 100 units transfused compared to 1.7–3.9 antibodies per 100 units transfused with ABO and RhD matching alone.17 The persistence of Rh alloimmunization despite Rh and K antigen matching in this multi-institutional study suggests the need for additional measures. RH genotyping can identify patients with partial antigens, and for some, donor selection can be modified to minimize foreign antigen exposure. For example, the most common partial C antigens in patients with SCD result from inheritance of the hybrid RHD*DIIIa-CE(4–7)-D (RHD*03N.01) or RHCE*CeRN (RHCE*02.10.01) alleles; patients with these alleles who lack a conventional C antigen are better served with C-negative units to prevent anti-C formation.7,18 Since recruitment of Black donors is necessary to support CEK matching programs as they are more likely to be C, E, and K-negative (44% vs 2% Whites), RH genotype matching of donors and patients may enhance use of these valuable donors.
This study has limitations, including its retrospective nature and variations in population size between sites. It is possible that some antibodies detected may represent autoantibodies, but to determine this is not feasible for a retrospective study. The majority of all and Rh alloantibodies identified were from patients at one institution since most patients (50.6%) were from CHOP, accounting for 62,032 (68.9%) total transfusions. Despite this, the total and Rh alloimmunization incidence was similar between CHOP, CHOA and SJCRH. CHOP is the only site that, for nearly 30 years, has been providing primarily Black donor units. However, there is increasing national attention to recruit more Black donors, particularly at blood centers that serve large numbers of patients with SCD. The similar alloimmunization rates seen at CHOA and SJCRH may suggest that patients at these sites are being exposed frequently to Black donors as well, although there was no specific donor program to recruit them.
This study has implications for improving transfusion practices to prevent Rh alloimmunization in SCD. Prophylactic RH matching has the potential to reduce Rh alloimmunization and its associated complications. The similar frequencies of variant RH among patients and Black donors suggests this could be feasible11 and would optimize the use of Black donor units. Future studies to minimize the cost of comprehensive RH genotyping, information technology solutions to electronically RH genotype match patients with donors, and real-world feasibility trials of RH genotype-matched red cell transfusions are needed prior to widespread implementation.
Supplementary Material
Acknowledgements
The authors thank patients and families who enrolled in the studies, blood donors, and members of the Blood Bank at CHOP, CHOA, SJCRH, UHRBCH and UCSF, and the Immunohematology and Genomics Laboratory at NYBC. We would like to thank Dr. Katharine Downes for providing details regarding serologic workup and methods at UHRBCH. This work was supported by the Doris Duke Charitable Foundation Innovations in Clinical Research Award (STC, CMW), NHLBI R01 HL147879 (STC, CMW, DFF), and NHLBI U01 HL134696 (STC, CMW). NI analyzed data and wrote the manuscript; ZZ analyzed the data; SV, DFF, SU performed the research and edited the manuscript; RF, MY, CP, SK, JH, YZ enrolled patients, provided patient specimens, and edited the manuscript; CMW and STC designed the research study, analyzed the data, and wrote the manuscript.
RMF serves on medical advisory boards for Global Blood Therapeutics, Forma Therapeutics, and Cerus, and receives research funding from Forma Therapeutics and Cerus. RMF also serves as a consultant for REDSIV-Pediatric which is funded by the NIH/NHLBI.
Funding:
This work was supported by the Doris Duke Charitable Foundation Innovations in Clinical Research Award (STC, CMW), the National Heart, Lung, and Blood Institute R01 HL147879 (STC, CMW, DFF), U01 HL134696 (STC, CMW).
Footnotes
Conflicts of interest:
The remaining authors have no conflicts to disclose.
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