Key Points

  • Rh serologic phenotype–matched transfusions from minority donors do not prevent all Rh alloimmunization in patients with SCD.

  • Variant RH genes are common in patients with SCD and contribute to Rh alloimmunization and transfusion reactions.

Abstract

Red blood cell (RBC) transfusion is a key treatment of patients with sickle cell disease (SCD) but remains complicated by RBC immunization. In the present study, we evaluated the effects of antigen matching for Rh D, C, and E, and K and transfusion from African American donors in 182 patients with SCD. Overall, 71 (58%) chronic and 9 (15%) episodically transfused patients were alloimmunized. Fifty-five (45%) chronic and 7 (12%) episodically transfused patients were Rh immunized. Of 146 antibodies identified, 91 were unexplained Rh antibodies, one-third of which were associated with laboratory evidence of delayed transfusion reactions. Fifty-six antibodies occurred in patients whose RBCs were phenotypically positive for the corresponding Rh antigen and 35 in patients whose RBCs lacked the antigen and were transfused with Rh-matched RBCs. High-resolution RH genotyping revealed variant alleles in 87% of individuals. These data describe the prevalence of Rh alloimmunization in patients with SCD transfused with phenotypic Rh-matched African American RBCs. Our results suggest that altered RH alleles in both the patients and in the donors contributed to Rh alloimmunization in this study. Whether RH genotyping of patients and minority donors will reduce Rh alloimmunization in SCD needs to be examined.

Introduction

The use of transfusion therapy for sickle cell disease (SCD) is increasing due to expanded indications, increased availability of erythrocytapheresis, and oral chelators to treat transfusional iron overload.1  However, alloimmunization to red blood cell (RBC) blood group antigens remains a major complication for patients with SCD and often presents significant challenges in their medical management.2,3  The incidence of alloimmunization in patients with SCD ranges from 7% to 47%, dependent on age, RBC exposures, and extent of antigen matching for blood groups other than ABO and RhD.4-12  An estimated 4% to 11% of patients with SCD who receive transfusions develop overt delayed hemolytic transfusion reactions (DHTRs),13-15  but mild DHTRs may be unrecognized.

Sensitization to Rh antigens (D, C, c, E, and e) and to K comprise the majority of the RBC antibodies encountered in SCD.4,5,9  One major explanation of the high rates of alloimmunization is the disparate distribution of RBC antigens between donors primarily of European ancestry and patients with SCD primarily of African ancestry.9  One strategy to decrease alloimmunization in SCD is provision of phenotype matched RBCs for C, E, and K antigens.8,10,12,16  Transfusion with units from African American donors has also been suggested,9,10  although an increase in production of antibodies to low incidence antigens present primarily in minority groups is predicted.17  Phenotype matching for additional minor antigens in the Kidd, Duffy, and MNS systems reveal that more stringent matching results in lower alloimmunization rates,6,18  but there is no standard of practice.19 

The Rh system is a complex blood group system and includes >50 different serologic specificities.20  The RH locus is comprised of 2 homologous genes, RHD and RHCE, which encode the D antigen and the CE antigens in various combinations (ce, cE, Ce, or CE), respectively. RHD and RHCE are inherited as haplotypes, and expression of the proteins is exclusive to erythrocytes. Genetic diversity of the RH locus has been revealed in the last decade, with >200 RHD and 80 RHCE alleles reported. These potentially encode variant or altered antigens due to amino acid changes in the Rh proteins.3,21,22  The RBCs may lack common Rh antigenic epitopes or carry novel epitopes. Standard RBC antigen typing does not distinguish the presence of Rh variants, which are more prevalent in individuals of African ancestry. For example, RHD encodes the D antigen (“Rh positive”), but individuals with altered RHD encoding partial D, defined as missing some D epitopes, may form anti-D when exposed to conventional D antigen.23-26  In contrast, individuals with altered RHD encoding weak D, defined as expressing a reduced amount of D antigen but not lacking epitopes, are not typically at risk for anti-D.20,27  Variant RHCE alleles are also prevalent in individuals of African descent, with alleles encoding partial e antigen most often encountered.24,28-32  A frequent variant allele in this ethnic group is the hybrid DIIIa-CE(4-7)-D gene, in which several RHCE exons have replaced the corresponding region of RHD.33  This allele encodes a partial C antigen and does not encode D antigen. RBCs are phenotypically C+, but individuals with this allele are at risk for anti-C when exposed to C antigen encoded by a conventional allele.29 

This study was undertaken to assess the effects of antigen matching for D, C, E, and K and transfusion primarily from African American donors on alloimmunization rates, antibody specificity, and clinical significance in pediatric and young adult patients with SCD. The association of inherited RH genotypes with antibody production in patients on chronic and episodic transfusion protocols was investigated.

Methods

Study design

This study is a 15-year retrospective analysis of patients with SCD at the Comprehensive Sickle Cell Center (CSCC) at the Children’s Hospital of Philadelphia (CHOP), transfused with RBCs that were prospectively matched for D, C, E, and K and were primarily from African American donors. Molecular analyses were performed between 2009 and 2012.

Transfusion protocol

Since 1994, the institutional policy for transfusion of patients with SCD has been to antigen match prospectively for D, C, E, and K antigens; patients whose RBCs lack the antigen are transfused with antigen-negative donor units. Efforts to provide ethnically matched RBC units began in 1997 through the “Blue Tag” Program consisting of donors who self-identify as African American and designate their donation to support children with SCD, although patients are not assigned to specific individual donor(s). Transfusions were mostly from African American donors, with some exceptions related to patient-specific antibodies rather than supply. These included patients with anti-D, for whom antigen-negative donors are primarily found in whites, and occasional patients alloimmunized to multiple antigens needing urgent transfusion. Patients received hemoglobin S-negative, leukocyte-reduced, and irradiated RBCs <21 days old. Patients on a chronic transfusion program had a target pretransfusion percent hemoglobin S of 30% or 50%, depending on the indication.

Patient population

In accordance with the Declaration of Helsinki, patients were enrolled after informed consent under a protocol approved by the Institutional Review Board at the Children’s Hospital of Philadelphia to review medical records and retain patient samples to study alloimmunization. Clinical records were reviewed from patient birth to June 1, 2012. By parental history, all patients were of African descent. Subjects were interviewed at enrollment to determine whether they received all their transfusion support at our facility. Additionally, because >99% of RBC units in this cohort were transfused on an outpatient basis every 3 to 5 weeks for chronic transfusion therapy, the patient history was reviewed for missed appointments or laboratory values reflecting possible off-program transfusion. With the exception of 1 patient, no alloimmunized patients were transfused outside our institution for the 12 months preceding new antibody detection.

Twenty-five of the 123 chronic and 7 of 59 episodically transfused patients received ≥1 transfusion prior to initiation of C, E, and K matching with African American donors. Twelve antibodies occurred in 9 patients before these 2 strategies were implemented: 3 anti-C, 2 anti-K, 3 anti-M, 1 anti-CW, 1 anti-Cob, 1 anti-Kna, 1 anti-Lea, and 3 anti-M (supplemental Table 1). We excluded these antibodies, but not the patients, because 23 were not alloimmunized, and thus were still at risk; the 9 with antibodies were at risk for additional antibodies. Of the 9 alloimmunized prior to 1997, 7 formed additional antibodies during the study period: 3 anti-D, 2 anti-C, 1 anti-E, 2 anti-Kpa, and 1 anti-S (supplemental Table 1).

Laboratory testing

Patient RBCs were serologically phenotyped before transfusion for ABO; Rh (D, C, c, E, e); Kell (K); Duffy (Fya, Fyb); Kidd (Jka, Jkb); Lewis (Lea, Leb); MNS (M, N, S, s); and P1. An antibody screen, complete blood count, and hemoglobin quantitation were performed prior to each transfusion. Antibody testing was performed with the low ionic strength saline tube technique until 2000 and with a gel-based method (Ortho Diagnostics) thereafter. We defined a warm autoantibody as the presence of a positive direct antiglobulin test with a panagglutinin in the serum or eluate with similar strength of reactivity to all cells tested with no apparent specificity.

RH high-resolution genotyping

Genomic DNA was extracted from peripheral blood (QIAamp; Qiagen, Valencia, CA), polymerase chain reaction (PCR) amplified, and analyzed by a combination of PCR-restriction fragment length polymorphism assays, as described previously.24  Samples were also tested with prototype RHD and RHCE BeadChip arrays (BioArray, Warren, NJ). For high-resolution genotyping, PCR was performed with RHD- and RHCE exon-specific primers in the flanking introns, directly sequenced, and compared with conventional RHD (GenBank accession no. L08429) or RHCE (GenBank accession no. DQ322275). To establish allelic associations, Rh-cDNA cloning and sequencing were performed. RNA was isolated from reticulocytes with TriZol (Invitrogen, Carlsbad, CA). Reverse transcription was performed with gene-specific primers (Superscript First Strand Synthesis; Invitrogen). PCR products were purified (ExoSAP-IT; USB, Cleveland, OH) and sequenced.

Determination of transfusion outcome and statistical analysis

Antibodies were recorded with detection date and RBC exposure number at the time of antibody development. We compared the percent hemoglobin S and hemoglobin levels at the time of new antibody detection to the patient’s baseline values. The baseline was calculated as the mean pretransfusion percent hemoglobin S and hemoglobin levels on each visit in the 6 to 12 months preceding the appearance of a specific antibody. The standard deviation (SD) from the mean was calculated, and antibodies associated with a difference in values >2 × SD of their individual mean (Z-score > 2.0, corresponding to two-tailed P < .05) were considered a clinically significant delayed transfusion reaction (DTR). The Student t test was used to compare parametric data between groups, and the Mann-Whitney test was used for nonparametric data. The χ2 test was used for categorical data. A two-tailed P < .05 was considered statistically significant.

Results

Subjects

The majority of subjects (91.2%) were homozygous HbSS, 5.0% were HbSC, 3.3% were HbSβ-thalassemia, and 0.5% were HbSO-Arab. A review of the transfusion history was performed for 182 patients with ≥1 RBC exposure (Table 1). Fifty-nine individuals (32.4%) had received transfusions episodically for acute complications of SCD or preoperative preparation. The mean number of exposures per patient was 4.6, the median was 3, and the range was 1 to 15 units. A total of 272 units were transfused to this group, primarily by simple transfusion. One hundred twenty-three patients (67.6%) had been or were currently managed with chronic transfusions and received 44 210 units by simple transfusion or erythrocytapheresis. Within this cohort, the mean number of exposures per patient was 354, the median was 230, and the range was 10 to 1460 units. The most common indications for chronic transfusion were primary and secondary stroke prevention (30.1% and 32.5%, respectively).

Table 1

Transfused patient characteristics

    Episodic category
 
Chronic category
 
 Overall demographics Episodic category Chronic category Alloimmunized Nonalloimmunized P value Alloimmunized Nonalloimmunized P value 
No. of patients 182         
Male/female 112/70 (61.5% male)         
Median age (years) 15.7 (range 0.5-41)         
Transfusion category          
 No. of patients (%)  59 (32.4) 123 (67.6)       
 Donor RBC exposures (units)          
  Mean  4.6 354       
  Median  230       
  Range  1-15 10-1460       
  Total for group  272 44 210       
Alloimmunization status          
 No. of patients (%)    9 (15.2) 50 (84.8) NA 71 (57.7) 52 (42.3) NA 
 No. of male patients (%)    3 (33.3) 28 (56.0) .317 48 (67.6) 30 (57.7) .003 
 Mean age (years)    14.5 11.6 .200 19.1 15.7 .007 
 Mean age at first transfusion (years)    3.9 5.3 .327 6.5 6.8 .755 
 Donor RBC exposures (units)          
  Mean    8.9 3.8 <.001 408.6 292.3 .045 
  Median    2.5 <.001 309 192.5 .008 
  Range    3-15 1-12 NA 12-1263 10-1460 NA 
  Total for group    80 192 NA 29 009 15 201 NA 
    Episodic category
 
Chronic category
 
 Overall demographics Episodic category Chronic category Alloimmunized Nonalloimmunized P value Alloimmunized Nonalloimmunized P value 
No. of patients 182         
Male/female 112/70 (61.5% male)         
Median age (years) 15.7 (range 0.5-41)         
Transfusion category          
 No. of patients (%)  59 (32.4) 123 (67.6)       
 Donor RBC exposures (units)          
  Mean  4.6 354       
  Median  230       
  Range  1-15 10-1460       
  Total for group  272 44 210       
Alloimmunization status          
 No. of patients (%)    9 (15.2) 50 (84.8) NA 71 (57.7) 52 (42.3) NA 
 No. of male patients (%)    3 (33.3) 28 (56.0) .317 48 (67.6) 30 (57.7) .003 
 Mean age (years)    14.5 11.6 .200 19.1 15.7 .007 
 Mean age at first transfusion (years)    3.9 5.3 .327 6.5 6.8 .755 
 Donor RBC exposures (units)          
  Mean    8.9 3.8 <.001 408.6 292.3 .045 
  Median    2.5 <.001 309 192.5 .008 
  Range    3-15 1-12 NA 12-1263 10-1460 NA 
  Total for group    80 192 NA 29 009 15 201 NA 

NA, not applicable; bold indicates statistically significant (P < .05).

RBC immunization in patients with SCD

Among 182 transfused patients, 9 of 59 episodic (15%) and 71 of 123 chronically transfused (58%) subjects were alloimmunized (Table 1). For episodically transfused patients, gender, age at data analysis, and age at first transfusion were not significantly different for alloimmunized compared with nonalloimmunized patients. However, alloimmunized patients received a greater number of RBC units compared with nonalloimmunized patients (8.9 vs 3.8 units, P < .001). In the chronically transfused cohort, male gender (P = .003), older age at data analysis (P = .007), and a greater mean number of RBC units (P = .045) were associated with alloimmunization (Table 1).

There were 146 specific antibodies identified in the serum of 80 individuals (Figure 1A). Among the 123 chronically transfused patients, 35 had 1 antibody, 21 had 2 antibodies, 9 had 3 antibodies, and 6 had >3 antibodies (Figure 1B). In the 59 episodically transfused patients, 6 had 1 antibody, 1 had 2 antibodies, and 2 had 3 antibodies (Figure 1B). Antibodies to low incidence antigens found primarily on RBCs of African American donors and predicted to be increased in patients receiving transfusion with minority donor units included 3 examples of anti-Goa, 2 anti-V/VS, and 5 anti-Jsa. Commonly encountered antibodies included 7 anti-M, 5 anti-S, and 4 anti-Fya. Antibodies to other low prevalence antigens included 4 anti-Kpa, 1 anti-CW, 2 anti-Sc2, and in the MNS system, 1 anti-Vw and 2 anti-He. Fifty percent of chronically transfused patients had a warm autoantibody compared with 5% of episodically transfused individuals (data not shown).

Figure 1

RBC immunization in patients with SCD transfused with Rh D, C, and E and K matched RBCs from minority donors. (A) One hundred forty-six specific antibodies in 123 chronically and 59 episodically transfused patients. (B) Number of antibodies per patient. (C) Number of Rh antibodies per patient. (D) Number of patients with anti-D, -C, -E, or -e in their serum and whose RBCs type positive or negative for the antigen.

Figure 1

RBC immunization in patients with SCD transfused with Rh D, C, and E and K matched RBCs from minority donors. (A) One hundred forty-six specific antibodies in 123 chronically and 59 episodically transfused patients. (B) Number of antibodies per patient. (C) Number of Rh antibodies per patient. (D) Number of patients with anti-D, -C, -E, or -e in their serum and whose RBCs type positive or negative for the antigen.

Notably, 94 of the 146 antibodies had specificity for common Rh antigens (D, C, E, e), and comprised nearly two-thirds (64.4%) of all antibodies (Figure 1A). Fifty-five (45%) chronically and 7 (12%) episodically transfused patients were Rh alloimmunized despite prophylactic Rh antigen matching, and 25 (40%) had >1 Rh antibody (Figure 1C). Only three cases of anti-E could be explained by transfusion of E+ RBCs to E− patients because they had made anti-e. Patient interviews excluded transfusion not matched for C, E, K antigens outside our institution (except 1 case), and look-back of donor center records and repeat antigen typing of donors when possible excluded labeling and antigen typing errors. Anti-D was identified in the serum of 30 patients (29 whose RBCs typed D+), anti-C in 29 (9 whose RBCs typed C+), anti-E in 19 (2 whose RBCs typed E+) and anti-e in 16 (all whose RBCs typed e+; Figure 1D). In summary, there were 56 unexplained Rh specificities identified in 45 patients whose RBCs typed positive for the corresponding antigen and 35 unexplained Rh specificities in 33 patients whose RBCs typed negative for the antigen and had received antigen-negative units. The Rh specificities had clear preference for antigen-positive cells and were distinguishable from warm autoantibodies.

Clinical significance of unexplained Rh antibodies

One overt hemolytic transfusion reaction requiring hospitalization was associated with anti-D in a D+ patient (Table 2, ID 292). DTRs are underreported in this patient group but are typically accompanied by an increased percent hemoglobin S level and/or a decrease in the hemoglobin/hematocrit.1  We evaluated whether patients experienced a DTR at the time of Rh antibody production by comparing the percent hemoglobin S and hemoglobin level with the patient’s baseline values (n = 82 evaluable occurrences in 56 patients). Figure 2 depicts hematologic data consistent with DTRs for 3 representative patients receiving chronic transfusions who made unexplained anti-D, -C, or -e. Twelve of 29 anti-D, 3 of 9 anti-C, and 5 of 16 anti-e occurring in D+, C+, and e+ individuals, respectively, were associated with a DTR (Table 2). Overall, 20 of 50 (40%) Rh antibodies evaluated in individuals positive for the corresponding antigen and 8 of 29 (28%) in antigen-negative individuals who received antigen-negative blood (Table 2) were associated with a DTR. One of 3 anti-E in E− patients who received E+ RBCs for management of anti-e also had a DTR (Table 2).

Table 2

Twenty-nine Rh antibodies in 25 patients associated with a significant change in hematologic parameters

Antibody specificity Concurrent Antibodies ID Hemoglobin S (%) Hemoglobin (g/dL) 
Baseline At antibody detection Z-score* Baseline At antibody detection Z-score* 
Patients whose RBCs type positive for the corresponding antigen         
 D (n = 12)  292† — 81.7 — 6.5 4.2 5.7 
E 95 27.1 68.0 10.4 9.1 7.5 3.2 
 41 25.9 38.2 5.9 9.4 8.9 2.4 
 78 28.8 45.1 5.4 10.2 9.7 −0.5 
C 138 19.3 61.6 4.3 10.4 8.2 4.0 
C 77 28.8 45.3 3.5 8.0 8.1 0.2 
 110 27.6 — — 10.6 7.9 2.7 
 50 44.3 58.8 1.6 8.5 6.4 2.8 
 99 29.6 45.7 2.5 8.9 8.2 −1.0 
 34 18.2 23.3 2.2 8.8 8.7 −0.2 
 145 26.0 32.7 2.1 9.5 9.5 −0.1 
 100 24.8 40.6 2.0 8.9 7.5 −1.8 
 C (n = 3)  95 25.3 31.2 1.0 9.3 5.0 6.9 
D 138 19.3 61.6 4.3 10.4 8.2 4.0 
 85 28.9 44.0 2.7 9.8 8.8 2.3 
 e (n = 5)  65 29.0 43.6 4.9 8.7 8.2 −0.7 
 108 23.1 50.7 4.4 7.6 6.3 4.1 
C 97† — 76.3 — 9.0 6.3 7.6 
 100 26.4 40.6 2.1 8.6 8.1 2.7 
 86 — 89.5 — 7.7 6.9 2.1 
Patients whose RBCs type negative for the corresponding antigen and received antigen negative RBCs         
 C (n = 4)  28 25.1 45.8 8.0 9.4 8.7 −1.3 
D 77 28.8 45.3 3.5 8.0 8.1 0.2 
e 97† — 76.3 — 9.0 6.3 7.6 
 130 37.5 34.9 −0.8 8.9 8.2 5.0 
 E (n = 4) D 95 27.1 68.0 10.4 9.1 7.5 3.2 
 103 24.3 66.1 8.9 10.0 7.6 6.9 
 159 50 — — 9.2 5.1 6.8 
Jkb 31 43.1 91.5 2.0 7.9 6.8 −1.1 
Patients whose RBCs type negative for the corresponding antigen and received antigen positive RBCs         
 E (n = 1)  63‡ 32.3 50.7 4.4 9.5 8.9 2.1 
Antibody specificity Concurrent Antibodies ID Hemoglobin S (%) Hemoglobin (g/dL) 
Baseline At antibody detection Z-score* Baseline At antibody detection Z-score* 
Patients whose RBCs type positive for the corresponding antigen         
 D (n = 12)  292† — 81.7 — 6.5 4.2 5.7 
E 95 27.1 68.0 10.4 9.1 7.5 3.2 
 41 25.9 38.2 5.9 9.4 8.9 2.4 
 78 28.8 45.1 5.4 10.2 9.7 −0.5 
C 138 19.3 61.6 4.3 10.4 8.2 4.0 
C 77 28.8 45.3 3.5 8.0 8.1 0.2 
 110 27.6 — — 10.6 7.9 2.7 
 50 44.3 58.8 1.6 8.5 6.4 2.8 
 99 29.6 45.7 2.5 8.9 8.2 −1.0 
 34 18.2 23.3 2.2 8.8 8.7 −0.2 
 145 26.0 32.7 2.1 9.5 9.5 −0.1 
 100 24.8 40.6 2.0 8.9 7.5 −1.8 
 C (n = 3)  95 25.3 31.2 1.0 9.3 5.0 6.9 
D 138 19.3 61.6 4.3 10.4 8.2 4.0 
 85 28.9 44.0 2.7 9.8 8.8 2.3 
 e (n = 5)  65 29.0 43.6 4.9 8.7 8.2 −0.7 
 108 23.1 50.7 4.4 7.6 6.3 4.1 
C 97† — 76.3 — 9.0 6.3 7.6 
 100 26.4 40.6 2.1 8.6 8.1 2.7 
 86 — 89.5 — 7.7 6.9 2.1 
Patients whose RBCs type negative for the corresponding antigen and received antigen negative RBCs         
 C (n = 4)  28 25.1 45.8 8.0 9.4 8.7 −1.3 
D 77 28.8 45.3 3.5 8.0 8.1 0.2 
e 97† — 76.3 — 9.0 6.3 7.6 
 130 37.5 34.9 −0.8 8.9 8.2 5.0 
 E (n = 4) D 95 27.1 68.0 10.4 9.1 7.5 3.2 
 103 24.3 66.1 8.9 10.0 7.6 6.9 
 159 50 — — 9.2 5.1 6.8 
Jkb 31 43.1 91.5 2.0 7.9 6.8 −1.1 
Patients whose RBCs type negative for the corresponding antigen and received antigen positive RBCs         
 E (n = 1)  63‡ 32.3 50.7 4.4 9.5 8.9 2.1 

For each antibody, the percent hemoglobin S and hemoglobin level at the time of first detection is indicated and compared with the patient’s individual baseline value (see Methods). For antibody specificity, the second entry indicates additional antibodies detected concurrently. —, data not available.

*

Z-score >2.0 for hemoglobin S level or <−2.0 for hemoglobin level correlates with P < .05, indicated in bold. ID in bold indicates patients with >1 Rh antibody detected concurrently.

Episodically transfused.

E− transfused with E+ RBCs for management of anti-e.

Figure 2

DTRs in patients with unexplained Rh antibodies. Percent hemoglobin S and hemoglobin levels in 3 representative cases of chronically transfused patients with anti-D, -C, and -e (see Table 2 for values of all DTRs associated with Rh antibodies). Each point represents a pretransfusion value coinciding with transfusions occurring at 3-week intervals. Arrows indicate time of antibody detection. The dotted line represents the mean percent hemoglobin S or hemoglobin level for that individual determined pretransfusion for 9 visits preceding antibody detection. The SD for means was calculated. The difference between the baseline and the value at time of antibody formation is expressed as a multiple of this SD, or Z-score. The gray shaded area indicates values that would have a Z-score <2. Z-score >2.0 for hemoglobin S level or <−2.0 for hemoglobin level correlates with P < .05. The double lines on the top charts indicate a several-month period when transfusions were discontinued.

Figure 2

DTRs in patients with unexplained Rh antibodies. Percent hemoglobin S and hemoglobin levels in 3 representative cases of chronically transfused patients with anti-D, -C, and -e (see Table 2 for values of all DTRs associated with Rh antibodies). Each point represents a pretransfusion value coinciding with transfusions occurring at 3-week intervals. Arrows indicate time of antibody detection. The dotted line represents the mean percent hemoglobin S or hemoglobin level for that individual determined pretransfusion for 9 visits preceding antibody detection. The SD for means was calculated. The difference between the baseline and the value at time of antibody formation is expressed as a multiple of this SD, or Z-score. The gray shaded area indicates values that would have a Z-score <2. Z-score >2.0 for hemoglobin S level or <−2.0 for hemoglobin level correlates with P < .05. The double lines on the top charts indicate a several-month period when transfusions were discontinued.

RH genetic diversity in patients with SCD

High-resolution analysis of RHD and RHCE was performed to determine RH diversity and allele prevalence in 226 patients with SCD and whether Rh immunization in the 182 transfused patients was associated with specific RH genotypes. Thirteen different RHD, 14 RHCE*ce, and 1 RHCE*Ce alleles encoding amino acid changes were present (Figure 3). Patients were homozygous, heterozygous, or compound heterozygous for variant RH alleles. At the RHD locus, there were 85 alleles encoding the recessive D negative (“Rh negative”) phenotype: 52 with a gene deletion, 12 with a 37-bp inactivating insertion (RHDψ), and 21 with a RHD-CE-D hybrid locus [DIIIa-CE(4-7)-D]. Of 367 RHD alleles encoding D antigen (“Rh positive”), 235 alleles were conventional sequence (64%) and 132 encoded RhD proteins with amino acid changes (36%). RHD*DAU0 was common (16% of alleles) and RHD*weak partial D 4.0 was relatively frequent (5%).

Figure 3

RHD and RHCE diversity in 226 patients with SCD.RH alleles identified in patients with SCD. Each gray box represents 1 of 10 exons in the RH genes. Black boxes represent exon exchange between RHD and RHCE. Vertical black lines indicate position in the exon encoding amino acid substitutions in the protein. Dashed lines indicate gene deletion. Arrowhead indicates 37-bp duplication. Hatched boxes represent exons encoding a frameshift and untranslated region of the inactive RHD pseudogene.

Figure 3

RHD and RHCE diversity in 226 patients with SCD.RH alleles identified in patients with SCD. Each gray box represents 1 of 10 exons in the RH genes. Black boxes represent exon exchange between RHD and RHCE. Vertical black lines indicate position in the exon encoding amino acid substitutions in the protein. Dashed lines indicate gene deletion. Arrowhead indicates 37-bp duplication. Hatched boxes represent exons encoding a frameshift and untranslated region of the inactive RHD pseudogene.

At the RHCE locus, the majority of alleles in African Americans are RHCE*ce encoding a C−c+ and E−e+ RBC phenotype.20  Among 358 RHCE*ce alleles, 100 were conventional (28%) and 258 were variant (72%). Most frequent variant RHCEs were RHCE*ce(48C) (19%), RHCE*ce(733G) (14%), RHCE*ce(48C,733G) (6%), RHCE*ce(254G) (6%), and RHCE*ceS (6%) (Figure 3). Among 52 RHCE*Ce, 1 variant designated RHCE*CeRN was present. Forty-two alleles were RHCE*cE. No RHCE*CE was identified.

More than one-third of RHD and more than one-half of RHCE alleles differed from the conventional sequence. In total, ≥1 nonconventional RH allele was identified in 86.7% (196/226), and 47.3% of individuals had ≥1 variant RHD and 1 variant RHCE allele.

Examination of Rh antibodies with RH genotypes for antigen-positive patients

We examined each Rh antibody and the patient’s RH genotype, clinical outcome, exposure number, and duration of antibody reactivity (Tables 3 and 4). Anti-D was identified in the serum of 29 D+ patients (Table 3). Seven individuals had only variant RHD alleles encoding amino acid changes. Anti-D was associated with an overt hemolytic reaction in 1 patient homozygous for DAU4 (ID 292), and DTRs occurred in 1 patient with DAU0/DAU5 (ID 50) and 1 with DAU0 only (ID 34) (DTR details, Table 2). The remaining 22 patients with anti-D had 1 or even 2 conventional RHD alleles; 9 cases were associated with a DTR. Eight patients had other antibodies concurrent with anti-D (7 unexplained anti-C or anti-E and 1 anti-S). There was no significant difference in DTR incidence between D+ patients lacking conventional RHD and those with ≥1 conventional RHD allele (P = .690). The mean RBC exposures at time of anti-D detection was also not significantly different between those 2 groups (P = .108) and ranged from 2 to 733 units. After anti-D detection, all patients received D− RBCs. Anti-D duration was not significantly different based on the presence of ≥1 conventional RHD (P = .946) or DTR occurrence (P = .473), and most were undetectable after 1 to 3 months. In contrast, anti-D in the onlyD− patient persists 9 years after initial detection (Table 3).

Table 3

RHD genotype and production of anti-D: clinical significance, donor exposures, and antibody duration

Antibody specificity Concurrent Antibodies ID RH genotype DTR RBC exposures Antibody demon-stration (months) 
RHD RHCE 
D+ patients lacking conventional RHD          
 D (n = 7)  292* DAU4 DAU4 ce(48C) ce(48C) Yes — 
 50 DAU0 DAU5 ce(48C) ce(48C) Yes 91 
 34 DAU0 Deleted D ce(733G) ce Yes 269 
104 DAU3 DAU5 ce(48C) cE No 39 19 
214* DAU0 Weak partial 4.0 ce(48C) ce(48C,733G) SS — 
 117 Weak partial 4.0 Weak partial 4.0 ce(254G) ce(48C,733G) No 32 
 14 DIVa-2 DIIIa-CE(4-7)-D ce ceS No 181 
D+ patients with one or more conventional RHD          
 D (n = 22)  100 RHD DIIIa-CE(4-7)-D ce(733G) ceS Yes 98 14 
 141* RHD DIIIa-CE(4-7)-D ce(733G) ceS No 36 
44 RHD DIIIa-CE(4-7)-D ce ceS No 116 
 94 RHD DIIIa-CE(4-7)-D ce ceS No 82 16 
138 RHD DIIIa-CE(4-7)-D Ce ceS Yes 103 5+ 
 110 RHD Weak partial 4.0 Ce ceS Yes 28 
 72 RHD DIVa-2 ceTI ceTI No 111 
28 RHD DAU0 ceHAR ce(48C) No 212 
 30 RHD DAU0 Ce ce(48C) No 177 
 99 RHD Deleted D Ce cE Yes 28 
 17 RHD Deleted D cE ce No 331 
 95 RHD Deleted D Ce ceTI No 155 
E 95 RHD Deleted D Ce ceTI Yes 369 1 
 21 RHD Deleted D Ce ce(733G) No 426 
 78 RHD Deleted D ce(733G) ce(254G) Yes 199 
 19 RHD RHD ce ce No 733 
 18 RHD RHD cE ce No 64 54 
 41 RHD RHD ce ce(733G) Yes 429 
 145 RHD RHD Ce ce(733G) Yes 48 
20 RHD RHD ce ce(733G) No 224 
 69 RHD RHD ce(733G) ce(733G) No 230 
77 RHD RHD ce ce(254G) Yes 12 
 103 RHD RHD ce(48C) ceS — — — 
D− patient          
 D  10 Deleted D Inactive RHD ψ ce(254G) ce(48C) No 314 112+ 
Antibody specificity Concurrent Antibodies ID RH genotype DTR RBC exposures Antibody demon-stration (months) 
RHD RHCE 
D+ patients lacking conventional RHD          
 D (n = 7)  292* DAU4 DAU4 ce(48C) ce(48C) Yes — 
 50 DAU0 DAU5 ce(48C) ce(48C) Yes 91 
 34 DAU0 Deleted D ce(733G) ce Yes 269 
104 DAU3 DAU5 ce(48C) cE No 39 19 
214* DAU0 Weak partial 4.0 ce(48C) ce(48C,733G) SS — 
 117 Weak partial 4.0 Weak partial 4.0 ce(254G) ce(48C,733G) No 32 
 14 DIVa-2 DIIIa-CE(4-7)-D ce ceS No 181 
D+ patients with one or more conventional RHD          
 D (n = 22)  100 RHD DIIIa-CE(4-7)-D ce(733G) ceS Yes 98 14 
 141* RHD DIIIa-CE(4-7)-D ce(733G) ceS No 36 
44 RHD DIIIa-CE(4-7)-D ce ceS No 116 
 94 RHD DIIIa-CE(4-7)-D ce ceS No 82 16 
138 RHD DIIIa-CE(4-7)-D Ce ceS Yes 103 5+ 
 110 RHD Weak partial 4.0 Ce ceS Yes 28 
 72 RHD DIVa-2 ceTI ceTI No 111 
28 RHD DAU0 ceHAR ce(48C) No 212 
 30 RHD DAU0 Ce ce(48C) No 177 
 99 RHD Deleted D Ce cE Yes 28 
 17 RHD Deleted D cE ce No 331 
 95 RHD Deleted D Ce ceTI No 155 
E 95 RHD Deleted D Ce ceTI Yes 369 1 
 21 RHD Deleted D Ce ce(733G) No 426 
 78 RHD Deleted D ce(733G) ce(254G) Yes 199 
 19 RHD RHD ce ce No 733 
 18 RHD RHD cE ce No 64 54 
 41 RHD RHD ce ce(733G) Yes 429 
 145 RHD RHD Ce ce(733G) Yes 48 
20 RHD RHD ce ce(733G) No 224 
 69 RHD RHD ce(733G) ce(733G) No 230 
77 RHD RHD ce ce(254G) Yes 12 
 103 RHD RHD ce(48C) ceS — — — 
D− patient          
 D  10 Deleted D Inactive RHD ψ ce(254G) ce(48C) No 314 112+ 

DTR, DTR from Table 2; RBC exposures, cumulative number of RBC units transfused prior to antibody detection; antibody demonstration, number of months anti-D was detected. Additional antibody detected concurrently is indicated in the second column. SS, confounded by splenic sequestration; —, data not available; +, remains detectable; italicized patient demonstrates anti-D recurrence.

*

Episodically transfused.

Table 4

RHCE genotype and production of anti-C, -e, or -E: clinical significance, donor exposures, and antibody duration

Antibody specificity Concurrent Antibodies ID RH genotype DTR RBC exposures Antibody demon-stration (months) 
RHCE RHD 
C+ patients lacking conventional RHCE*Ce          
 C (n = 5)  100 ceS ce(733G) DIIIa-CE(4-7)-D RHD No 52 39 
 94 ceS ce DIIIa-CE(4-7)-D RHD No 115 
 105* ceS cE DIIIa-CE(4-7)-D RHD No 12 — 
 85 ceS ceTI DIIIa-CE(4-7)-D DIVa-2 Yes 65 13 
 118 ceS ceTI DIIIa-CE(4-7)-D DIVa-3 — — — 
C+ patients with one conventional RHCE*Ce          
 C (n = 4) 138 Ce ceS RHD DIIIa-CE(4-7)-D Yes 103 
 95 Ce ceTI RHD Deleted D Yes 365 
 96 Ce ce(733G) RHD Deleted D No 82 32 
 21 Ce ce(733G) RHD Deleted D — — — 
e+ patients lacking conventional RHCE*ce          
 e (n = 14) 97* ce(48C) ce(48C) RHD RHD Yes — 
 75 ce(48C) ce(48C) DAU0 DAU5 No 91 
 108 ce(48C) ce(733G) DAU0 Inactive RHD ψ Yes 107 
 102 ce(48C) ce(733G) DAU0 RHD No 295 24 
 148 ce(48C) ce(733G) Weak partial 4.0 Weak partial 4.0 No 
 65 ce(48C) ceS DAU3 DIIIa Yes 174 19 
 63 ce(48C) ceS DAU0 DIIIa-CE(4-7)-D No 195 
 100 ce(733G) ceS RHD DIIIa-CE(4-7)-D Yes 62 36 
 96 ce(733G) Ce Deleted D RHD No 64 10 
 93 ce(733G) Ce RHD RHD No 24 
 45 ce(733G) cE RHD RHD — — — 
 86 ce(48C,733G) cE Weak partial 4.0 RHD Yes 
98 ce(254G) cE RHD Deleted D No 92 11 
60 ce(254G) cE DAU0 RHD No 40 22 
e+ patients with one conventional RHCE*ce          
 e (n = 2)  27 ce ce(48C) RHD DAU0 No 396 49 
 94 ce ceS RHD DIIIa-CE(4-7)-D No 281 
E+ patients with one conventional RHCE*cE          
 E (n = 2)  17 cE ce RHD Deleted D — 279 
104 cE ce(48C) DAU3 DAU5 No 39 19 
Antibody specificity Concurrent Antibodies ID RH genotype DTR RBC exposures Antibody demon-stration (months) 
RHCE RHD 
C+ patients lacking conventional RHCE*Ce          
 C (n = 5)  100 ceS ce(733G) DIIIa-CE(4-7)-D RHD No 52 39 
 94 ceS ce DIIIa-CE(4-7)-D RHD No 115 
 105* ceS cE DIIIa-CE(4-7)-D RHD No 12 — 
 85 ceS ceTI DIIIa-CE(4-7)-D DIVa-2 Yes 65 13 
 118 ceS ceTI DIIIa-CE(4-7)-D DIVa-3 — — — 
C+ patients with one conventional RHCE*Ce          
 C (n = 4) 138 Ce ceS RHD DIIIa-CE(4-7)-D Yes 103 
 95 Ce ceTI RHD Deleted D Yes 365 
 96 Ce ce(733G) RHD Deleted D No 82 32 
 21 Ce ce(733G) RHD Deleted D — — — 
e+ patients lacking conventional RHCE*ce          
 e (n = 14) 97* ce(48C) ce(48C) RHD RHD Yes — 
 75 ce(48C) ce(48C) DAU0 DAU5 No 91 
 108 ce(48C) ce(733G) DAU0 Inactive RHD ψ Yes 107 
 102 ce(48C) ce(733G) DAU0 RHD No 295 24 
 148 ce(48C) ce(733G) Weak partial 4.0 Weak partial 4.0 No 
 65 ce(48C) ceS DAU3 DIIIa Yes 174 19 
 63 ce(48C) ceS DAU0 DIIIa-CE(4-7)-D No 195 
 100 ce(733G) ceS RHD DIIIa-CE(4-7)-D Yes 62 36 
 96 ce(733G) Ce Deleted D RHD No 64 10 
 93 ce(733G) Ce RHD RHD No 24 
 45 ce(733G) cE RHD RHD — — — 
 86 ce(48C,733G) cE Weak partial 4.0 RHD Yes 
98 ce(254G) cE RHD Deleted D No 92 11 
60 ce(254G) cE DAU0 RHD No 40 22 
e+ patients with one conventional RHCE*ce          
 e (n = 2)  27 ce ce(48C) RHD DAU0 No 396 49 
 94 ce ceS RHD DIIIa-CE(4-7)-D No 281 
E+ patients with one conventional RHCE*cE          
 E (n = 2)  17 cE ce RHD Deleted D — 279 
104 cE ce(48C) DAU3 DAU5 No 39 19 

DTR, DTR from Table 2; RBC exposures, cumulative number of units transfused prior to antibody detection; antibody demonstration, the number of months antibody was detected. Additional antibody detected concurrently is indicated in the second column. —, data not available.

*

Episodically transfused patient.

Anti-C was identified in the serum of 9 C+ patients (Table 4). Five patients had partial C antigen encoded by a hybrid DIIIa-CE(4-7)-D (at the RHD locus), and 1 had a DTR. Of 4 patients with 1 conventional RHCE*Ce (3 were siblings), 2 had DTRs, and anti-D was also present in 1. DTR occurrence was not significantly different for patients with the hybrid DIIIa-CE(4-7)-D compared with those with 1 conventional RHCE*Ce (P = .270), but patient numbers were small. The mean number of exposures at time of anti-C detection was also not significantly different between the 2 groups (P = .229). After detection, patients received C− RBCs, and anti-C duration was not different based on the presence of 1 conventional RHCE*Ce (P = .400) or DTR occurrence (P = .200).

Anti-e was identified in the serum of 16 e+ patients (Table 4). Fourteen lacked a conventional RHCE*ce: 8 were homozygous for variant RHCE*ce, 2 had RHCE*Ce in trans, and 4 had RHCE*cE in trans. Anti-e was associated with a DTR in 5 of these 14 patients, and 3 also had anti-C. Two e+ patients with anti-e had a conventional RHCE*ce in trans to an altered RHCE*ce allele. The mean exposures at time of anti-e detection were 89 units for e+ patients lacking conventional RHCE*ce and 339 for those with 1 conventional RHCE*ce, which was significantly different despite the small number in the latter group (P = .002). The duration of anti-e demonstration was not significantly different from those with 1 conventional RHCE*ce allele (P = .690) or DTR occurrence (P = .942). After anti-e detection, patients received e− (E+) RBCs, and antibody demonstration ranged between 1 and 49 months. Three E−e+ individuals subsequently developed anti-E; only 1 was associated with a DTR (Table 2, ID 63).

Anti-E was identified in the serum of two E+ patients (Table 4). Both had conventional RHCE*cE, in trans to RHCE*ce or RHCE*ce(48C), respectively. The latter also had anti-D.

Incidence of Rh alloimmunization and related RH genotypes

We determined the incidence of Rh alloimmunization in 123 chronically transfused patients with only variant RH alleles, variant and conventional alleles, or only conventional alleles (supplemental Table 2). Of 117 D+ patients, anti-D was detected in 5 of 29 (17%) who inherited only variant RHD, 8 of 31 (26%) with 1 conventional and 1 variant RHD, and 13 of 57 (23%) with conventional RHD only (P = .720). Of 36 C+ patients, anti-C was detected in 4 of 10 (40%) individuals with only the hybrid RHD*DIIIa-CE(4-7)-D encoding partial C, 1 of 1 with conventional RHCE*Ce and hybrid RHD*DIIIa-CE(4-7)-D, and 3 of 25 (12%) with conventional RHCE*Ce only (P = .033). All 123 chronic transfused patients were e+; anti-e was found in 13 of 69 (19%) with only variant RHCE*ce, 2 of 36 (6%) with 1 conventional and 1 altered RHCE*ce, and 0 of 18 patients with only conventional RHCE*ce (P = .033). Of 19 E+ patients, anti-E was detected in 2 of 19 (11%) patients with conventional RHCE*cE.

One of 6 D− patients made anti-D (17%), 17 of 87 C− patients had anti-C (20%), and 13 of 104 E− patients had anti-E (13%), despite D-, C-, and E-negative transfusions, respectively.

Discussion

We report here the results of a 15-year experience of transfusing patients with SCD with donor units that were antigen matched for Rh D, C, and E, and K, and selected primarily from African American donors. A number of studies demonstrate that antigen matching for C, E, and K is associated with a decrease in antibodies,10,12,16  and extended phenotype matching to also include Jkb, Fya, and S can further minimize alloimmunization.5,6  The rationale for providing blood from ethnically similar donors is based on differences in RBC antigen frequency in white and black ethnic groups. For instance, the C−, E−, K−, Fya−, and Jkb− phenotype occurs in 26% of African Americans and 2% of whites. Based on antigen prevalence differences, it was hypothesized that alloimmunization would be reduced in patients with SCD by transfusion with blood selected from ethnically similar donors.34 

Indeed, antibodies to FY, JK, and S were rare, with a rate of 0.027/100 units. However, the overall incidence of alloimmunization was higher than expected, primarily due to a large number of unexpected Rh antibodies. The alloimmunization rate for patients on chronic transfusions was 0.30/100 units transfused compared with 0.055/100 units in 45 patients reported by Wahl et al11  and 0.11/100 units in 32 patients by Godfrey et al,35  all receiving chronic transfusions with C-, E-, and K-matched RBCs. These studies were also in pediatric and young adult patient populations, and the mean number of units transfused per patient was 243 and 137, respectively. A significant distinction in our study is the large number of unexpected Rh antibodies identified: 45% of chronic transfused patients and 12% of episodically transfused patients had antibodies to D, C, E, or e, and 40% had >1 Rh antibody, with a rate of 0.21/100 units for unexpected Rh antibodies and 0.09/100 for other blood groups. The main differences in the present study are the larger number of patients, the greater mean number of units transfused (354 units), and RBC transfusions primarily from African American donors.

In this study, 91 Rh antibodies were unexpected. Thirty-five occurred in antigen-negative patients receiving corresponding D−, C−, or E− RBCs who should not have made the antibody in the absence of exposure to antigen-positive RBCs. Fifty-six antibodies occurred in patients whose RBCs were positive for the antigen and should not have recognized the antigen as foreign and formed the antibody. Unexplained Rh antibodies in patients with SCD were occasionally observed in previous studies, including one anti-D in a D+ patient, one anti-C in a C− patient, and three anti-E in both E+ and E− patients, despite Rh matching.10,11,35  A recent study reported 5 patients with Rh variants discovered by RH genotyping after immunization to Rh antigens for which their RBCs were positive by serologic phenotype.12 

These Rh antibodies can be clinically significant, as one-third were associated with delayed transfusion reactions, defined by hematologic laboratory changes at antibody detection. Only a few patients presented with clinical symptoms of increased hemolysis or worsening anemia, but the majority had mild or no symptoms and did not seek medical attention. More data are needed to determine the true incidence of DTR and the clinical significance of Rh antibodies in this patient population. Routine laboratory evaluations that may provide evidence of DTRs should be monitored closely and specifically reviewed when patients develop new antibodies. Clinically significant Rh antibodies occurred after 1 RBC exposure to as many as several hundred units, suggesting that heavily transfused patients remain at risk, in contrast to a prior report that all clinically significant alloantibodies were detected within 6 months of initiating chronic transfusion therapy.10  Nearly all unexplained Rh antibodies were evanescent and not detected in the patient’s serum after several months, consistent with observations that many alloantibodies disappear within 6 months.36  However, anti-D in D− patients typically persist, lasting for many years,36  but >80% of anti-D in D+ patients in this study were no longer demonstrable after 1 to 6 months. In contrast, the one anti-D in a D− patient reported here was likely due to transfusion with an RBC unit that expressed a weak D antigen not detected by standard donor typing,20  and the anti-D remains detectable after 9 years.

High-resolution RH genotyping revealed that 87% of patients inherited ≥1 variant RH allele, demonstrating the tremendous RH diversity in this population. These variant alleles potentially encode altered or partial Rh antigens not distinguished from common antigens with routine serologic typing. Twenty-six Rh antibodies occurred in patients homozygous for variant RH alleles. Conversely, we observed an absence of anti-e in chronically transfused patients with only conventional RHCE*ce (supplemental Table 2). Correlating specific RH alleles or haplotypes with alloimmunization or DTR was not possible due to the small numbers of individuals with identical RHD and RHCE genotypes. Comparison of antibody production, DTR occurrence, exposure number, and antibody duration was performed broadly between patients with and without variant alleles, but a large multi-institutional study is necessary to address the risk of alloimmunization and DTR with specific RH haplotypes.

RH diversity in patients contributes to Rh alloimmunization, but 65 unexpected Rh antibodies were not explained by homozygosity for altered alleles at the patient’s corresponding RH loci. Thus, the role of minority donor RBCs requires study. We hypothesize that African American donors have the same degree of RH heterogeneity. Rh specificities in the serum of patients who typed negative for D, C, or E antigens (n = 35 cases) or who tested positive and carry conventional alleles (n = 30 cases) likely reflect an immune response to a foreign Rh complex on African American donor units. Rh epitopes and Rh antigen specificities are complex, may be cross-reactive with other Rh antigens, and are not always straightforward. RhD-like epitopes, reactive with anti-D, can be expressed on altered Rhce proteins,31,37  C-like epitopes on variant RhD and Rhce proteins,32,38  and E-like antigens on RhCe proteins.39  Thus, future studies will address whether RBCs from African American donors stimulate complex Rh specificities.

These findings suggest that RH diversity in patients with SCD necessitates an alternative approach to improve RBC matching. As blood group genotyping technology continues to expand beyond reference laboratories to donor centers and hospitals, and as costs further decline, molecular analysis to refine RBC matching for African American patients and donors should be feasible. Although RH genotype–matched RBCs may be supply or cost prohibitive for all patients, transfusion management can be tailored for specific patient genotypes. For example, our transfusion policy for C+ patients with the hybrid DIIIa-CE(4-7)-D that encodes a partial C antigen (and who lack conventional RHCE*Ce) is to provide C− RBCs prospectively.29  Another potential strategy is to provide D− RBCs to D+ patients whose RHD genotypes predict partial D expression only. However, because D− RBCs are much less frequent among African-American donors, such a policy may result in increased alloantibodies to other RBC antigens with disparate distribution between Europeans and Africans (Jkb, Fya, S).9  More extended matching to include those antigens might be considered to minimize alloimmunization risk5,6  but may be challenging to supply for patients requiring chronic erythrocytapheresis. Future studies to determine the clinical significance of specific RH genotypes are needed, as well as prospective trials to assess RBC matching strategies based on patient and/or donor blood group genotypes.

The major findings of this study are (1) transfusion of patients with SCD using African American donor units antigen matched for D, C, E, and K did not reduce Rh alloimmunization; (2) antibodies to low incidence antigens found primarily on RBCs of African American donors were not significantly increased, but rather a large number of unexplained Rh antibodies with apparent common specificities were found; (3) ∼30% of unexpected Rh antibodies were associated with laboratory evidence of delayed transfusion reactions; and (4) altered RH alleles were present in 87%, and some, but not all Rh antibodies, were explained by inheritance of altered RH. This study suggests that the presence of 1 conventional RH allele is not protective against alloimmunization and that RH diversity in African American patients and donors contributes to the high rate of Rh alloimmunization seen here. Future studies will aim to determine whether RH genotyping of patients and donors can guide RBC selection to prevent Rh alloimmunization.

The online version of this article contains a data supplement.

There is an Inside Blood commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

The authors thank the patients and families who enrolled in the study, the staff of the Comprehensive Sickle Cell Center and the Blood Bank at The Children’s Hospital of Philadelphia, and the members of the Immunohematology and Genomics Laboratory at the New York Blood Center and the American Red Cross Molecular Laboratory, Philadelphia, PA.

This work was supported in part by the Doris Duke Innovations in Clinical Research Award grant 2011097 (S.T.C. and C.M.W.).

Authorship

Contribution: S.T.C., D.F.F., and C.M.W. designed the study, analyzed results, and wrote the manuscript; T.J. and S.V. conducted research, analyzed results, and provided helpful discussions; and K.S.-W. analyzed results, formulated discussions, and assisted with the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Stella T. Chou, 3615 Civic Center Blvd, Abramson Research Center 316D, Philadelphia, PA 19104; e-mail: chous@email.chop.edu.

References

References
1
Smith-Whitley
K
Thompson
AA
Indications and complications of transfusions in sickle cell disease.
Pediatr Blood Cancer
2012
, vol. 
59
 
2
(pg. 
358
-
364
)
2
Chou
ST
Liem
RI
Thompson
AA
Challenges of alloimmunization in patients with haemoglobinopathies.
Br J Haematol
2012
, vol. 
159
 
4
(pg. 
394
-
404
)
3
Yazdanbakhsh
K
Ware
RE
Noizat-Pirenne
F
Red blood cell alloimmunization in sickle cell disease: pathophysiology, risk factors, and transfusion management.
Blood
2012
, vol. 
120
 
3
(pg. 
528
-
537
)
4
Aygun
B
Padmanabhan
S
Paley
C
Chandrasekaran
V
Clinical significance of RBC alloantibodies and autoantibodies in sickle cell patients who received transfusions.
Transfusion
2002
, vol. 
42
 
1
(pg. 
37
-
43
)
5
Castro
O
Sandler
SG
Houston-Yu
P
Rana
S
Predicting the effect of transfusing only phenotype-matched RBCs to patients with sickle cell disease: theoretical and practical implications.
Transfusion
2002
, vol. 
42
 
6
(pg. 
684
-
690
)
6
Lasalle-Williams
M
Nuss
R
Le
T
Cole
L
Hassell
K
Murphy
JR
Ambruso
DR
Extended red blood cell antigen matching for transfusions in sickle cell disease: a review of a 14-year experience from a single center (CME).
Transfusion
2011
, vol. 
51
 
8
(pg. 
1732
-
1739
)
7
Miller
ST
Kim
HY
Weiner
DL
, et al. 
Investigators of the Sickle Cell Disease Clinical Research Network (SCDCRN)
Red blood cell alloimmunization in sickle cell disease: prevalence in 2010.
Transfusion
2013
, vol. 
53
 
4
(pg. 
704
-
709
)
8
Sakhalkar
VS
Roberts
K
Hawthorne
LM
McCaskill
DM
Veillon
DM
Caldito
GC
Cotelingam
JD
Allosensitization in patients receiving multiple blood transfusions.
Ann N Y Acad Sci
2005
, vol. 
1054
 (pg. 
495
-
499
)
9
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
, vol. 
322
 
23
(pg. 
1617
-
1621
)
10
Vichinsky
EP
Luban
NL
Wright
E
Olivieri
N
Driscoll
C
Pegelow
CH
Adams
RJ
Stroke Prevention Trail in Sickle Cell Anemia
Prospective RBC phenotype matching in a stroke-prevention trial in sickle cell anemia: a multicenter transfusion trial.
Transfusion
2001
, vol. 
41
 
9
(pg. 
1086
-
1092
)
11
Wahl
SK
Garcia
A
Hagar
W
Gildengorin
G
Quirolo
K
Vichinsky
E
Lower alloimmunization rates in pediatric sickle cell patients on chronic erythrocytapheresis compared to chronic simple transfusions.
Transfusion
2012
, vol. 
52
 
12
(pg. 
2671
-
2676
)
12
O’Suoji
C
Liem
RI
Mack
AK
Kingsberry
P
Ramsey
G
Thompson
AA
Alloimmunization in sickle cell anemia in the era of extended red cell typing [published online ahead of print March 18, 2013].
Pediatr Blood Cancer
13
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
, vol. 
148
 
11
(pg. 
2485
-
2489
)
14
de Montalembert
M
Dumont
MD
Heilbronner
C
, et al. 
Delayed hemolytic transfusion reaction in children with sickle cell disease.
Haematologica
2011
, vol. 
96
 
6
(pg. 
801
-
807
)
15
Talano
JA
Hillery
CA
Gottschall
JL
Baylerian
DM
Scott
JP
Delayed hemolytic transfusion reaction/hyperhemolysis syndrome in children with sickle cell disease.
Pediatrics
2003
, vol. 
111
 
6 Pt 1
(pg. 
e661
-
e665
)
16
Venkateswaran
L
Teruya
J
Bustillos
C
Mahoney
D
Jr
Mueller
BU
Red cell exchange does not appear to increase the rate of allo- and auto-immunization in chronically transfused children with sickle cell disease.
Pediatr Blood Cancer
2011
, vol. 
57
 
2
(pg. 
294
-
296
)
17
Reid
ME
Denomme
GA
DNA-based methods in the immunohematology reference laboratory.
Transfus Apheresis Sci
2011
, vol. 
44
 
1
(pg. 
65
-
72
)
18
Tahhan
HR
Holbrook
CT
Braddy
LR
Brewer
LD
Christie
JD
Antigen-matched donor blood in the transfusion management of patients with sickle cell disease.
Transfusion
1994
, vol. 
34
 
7
(pg. 
562
-
569
)
19
Osby
M
Shulman
IA
Phenotype matching of donor red blood cell units for nonalloimmunized sickle cell disease patients: a survey of 1182 North American laboratories.
Arch Pathol Lab Med
2005
, vol. 
129
 
2
(pg. 
190
-
193
)
20
Chou
ST
Westhoff
CM
Roback
JD
The Rh system.
Technical Manual
2011
Bethesda, MD
American Association of Blood Banks
(pg. 
389
-
410
)
21
Chou
ST
Westhoff
CM
Molecular biology of the Rh system: clinical considerations for transfusion in sickle cell disease.
Hematology Am Soc Hematol Educ Program
2009
, vol. 
2009
 
1
(pg. 
178
-
184
)
22
Chou
ST
Westhoff
CM
The role of molecular immunohematology in sickle cell disease.
Transfus Apheresis Sci
2011
, vol. 
44
 
1
(pg. 
73
-
79
)
23
Wagner
FF
Ladewig
B
Angert
KS
Heymann
GA
Eicher
NI
Flegel
WA
The DAU allele cluster of the RHD gene.
Blood
2002
, vol. 
100
 
1
(pg. 
306
-
311
)
24
Westhoff
CM
Vege
S
Halter-Hipsky
C
Whorley
T
Hue-Roye
K
Lomas-Francis
C
Reid
ME
DIIIa and DIII Type 5 are encoded by the same allele and are associated with altered RHCE*ce alleles: clinical implications.
Transfusion
2010
, vol. 
50
 
6
(pg. 
1303
-
1311
)
25
Hemker
MB
Ligthart
PC
Berger
L
van Rhenen
DJ
van der Schoot
CE
Wijk
PA
DAR, a new RhD variant involving exons 4, 5, and 7, often in linkage with ceAR, a new Rhce variant frequently found in African blacks.
Blood
1999
, vol. 
94
 
12
(pg. 
4337
-
4342
)
26
Castilho
L
Rios
M
Rodrigues
A
Pellegrino
J
Jr
Saad
ST
Costa
FF
High frequency of partial DIIIa and DAR alleles found in sickle cell disease patients suggests increased risk of alloimmunization to RhD.
Transfus Med
2005
, vol. 
15
 
1
(pg. 
49
-
55
)
27
Wagner
FF
Gassner
C
Müller
TH
Schönitzer
D
Schunter
F
Flegel
WA
Molecular basis of weak D phenotypes.
Blood
1999
, vol. 
93
 
1
(pg. 
385
-
393
)
28
Noizat-Pirenne
F
Lee
K
Pennec
PY
, et al. 
Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety.
Blood
2002
, vol. 
100
 
12
(pg. 
4223
-
4231
)
29
Tournamille
C
Meunier-Costes
N
Costes
B
, et al. 
Partial C antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunization.
Transfusion
2010
, vol. 
50
 
1
(pg. 
13
-
19
)
30
Pham
BN
Peyrard
T
Juszczak
G
, et al. 
Analysis of RhCE variants among 806 individuals in France: considerations for transfusion safety, with emphasis on patients with sickle cell disease.
Transfusion
2011
, vol. 
51
 
6
(pg. 
1249
-
1260
)
31
Flegel
WA
Wagner
FF
Chen
Q
Schlanser
G
Frame
T
Westhoff
CM
Moulds
MK
The RHCE allele ceCF: the molecular basis of Crawford (RH43).
Transfusion
2006
, vol. 
46
 
8
(pg. 
1334
-
1342
)
32
Westhoff
CM
Vege
S
Halter Hipsky
C
, et al. 
RHCE*ceTI encodes partial c and partial e and is often in cis to RHD*DIVa.
Transfusion
2013
, vol. 
53
 
4
(pg. 
741
-
746
)
33
Singleton
BK
Green
CA
Avent
ND
, et al. 
The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype.
Blood
2000
, vol. 
95
 
1
(pg. 
12
-
18
)
34
Sosler
SD
Jilly
BJ
Saporito
C
Koshy
M
A simple, practical model for reducing alloimmunization in patients with sickle cell disease.
Am J Hematol
1993
, vol. 
43
 
2
(pg. 
103
-
106
)
35
Godfrey
GJ
Lockwood
W
Kong
M
Bertolone
S
Raj
A
Antibody development in pediatric sickle cell patients undergoing erythrocytapheresis.
Pediatr Blood Cancer
2010
, vol. 
55
 
6
(pg. 
1134
-
1137
)
36
Tormey
CA
Stack
G
The persistence and evanescence of blood group alloantibodies in men.
Transfusion
2009
, vol. 
49
 
3
(pg. 
505
-
512
)
37
Chen
Q
Hustinx
H
Flegel
WA
The RHCE allele ceSL: the second example for D antigen expression without D-specific amino acids.
Transfusion
2006
, vol. 
46
 
5
(pg. 
766
-
772
)
38
Hipsky
CH
Hue-Roye
K
Lomas-Francis
C
Huang
CH
Reid
ME
Molecular basis of the rare gene complex, DIVa(C)-, which encodes four low-prevalence antigens in the Rh blood group system.
Vox Sang
2012
, vol. 
102
 
2
(pg. 
167
-
170
)
39
Vege
S
Lomas-Francis
C
Hu
Z
Hue-Roye
K
Patel
P
Westhoff
CM
 
E antigen typing discrepancy reveals a novel 674C>G change (Ser225Cys) on RhCe responsible for expression of some E epitopes (S54-0301) [abstract]. Transfusion. 2012;52:34A