RBC Antibodies Research Articles

Vasoactive mediators and hypotensive transfusion reactions

Vasoactive mediators and hypotensive transfusion reactions

High additional maternal red cell alloimmunization after Rhesus- and K-matched intrauterine intravascular transfusions for hemolytic disease of the fetus

Intrauterine transfusion (IUT) is a life-saving therapy for the severely anemic fetus with hemolytic disease. However, maternal additional antibody formation is a complication of the procedure. In this study, we determined antibody formation after introduction of preventive Rh-D, -C, -c, -E, and -e and K matching of IUT donors. This was a retrospective follow-up study. During an 11-year period, 686 Rhesus- and K-matched IUTs were performed in 233 pregnancies and in 95% (652/686) posttransfusion antibody testing was performed after a median interval of 21 days. Twenty-five percent (53/212) of the women formed 64 new antibodies and, compared to our previous study, this incidence was not decreased by the use of Rhesus- and K-matched donors. After delivery, 72% (153/212) of the women had multiple RBC antibodies. Additional antibodies were in 48% (31/64) directed against Rhesus and K antigens, induced by the fetus, or as natural antibodies. In 52% (33/64) the antibodies were directed against non-Rhesus and -K antigens and in 65% (11/17) of eligible cases the IUT donor and not the fetus expressed the corresponding antigen(s). Despite Rhesus- and K-matching, women treated with IUTs still show strikingly broad red cell alloimmunization. More extensive IUT donor red cell matching, including FY, JK, and S antigens, to reduce the formation of new red cell antibodies should be explored.

Prevalence of HLA antibodies in transfused patients with and without red blood cell antibodies

BACKGROUND: Multiply transfused patients are at increased risk of developing red cell (RBC) antibodies, as well as antibodies to HLA. Although pretransfusion testing screens for RBC antibodies, no such testing is routinely performed for HLA antibodies. Determining which patients are more likely to make HLA antibodies may be important for patients undergoing elective surgery where platelets (PLTs) may be required. It is hypothesized that patients with RBC alloantibodies may be more likely to have HLA antibodies than previously transfused patients without RBC antibodies. STUDY DESIGN AND METHODS: Blood was collected from 53 adult male surgical patients with RBC alloantibodies and a control group of 69 similar male patients with a history of previous transfusions but no evidence of RBC alloimmunization. The samples were tested for the presence of immunoglobulin G Class I HLA antibodies by enzyme-linked immunosorbent assay. RESULTS: Of the 53 samples from patients with RBC alloantibodies, 12 (22.6%) also had HLA antibodies, whereas only 7 (10.1%) of the 69 patients in the control group had HLA antibodies (p < 0.03). CONCLUSIONS: There is a significant difference between the rates of HLA alloimmunization in male patients with RBC antibodies versus multiply transfused patients without RBC antibodies. Screening for HLA antibodies may be warranted in patients with RBC alloantibodies who might require PLT transfusion support for elective surgery.

Exportin 4, a Potential Amplifier of TGF-β Signaling, Is Increased in Primary Baboon Bone Marrow Erythroblasts Following Decitabine Treatment.

The DNA demethylating drug decitabine increased fetal hemoglobin (HbF) to therapeutic levels and reduced the level of DNA methylation of the γ-globin gene promoter in patients with sickle cell disease and in experimental baboons. Whether decreased DNA methylation of the γ-globin gene is solely responsible for increased HbF following decitabine treatment is unknown. Increased platelet counts in patients with sickle cell disease and myelodysplastic syndrome and in experimental baboons following decitabine treatment suggest that decitabine also affects hematopoietic differentiation. To investigate to what extent the mechanism responsible for the ability of decitabine to reactivate HbF and alter hematopoietic differentiation may involve the induction of other unknown genes, the global pattern of gene expression in purified primary bone marrow erythroblasts pre- and post-decitabine treament was analyzed. Baboons were phlebotomized for ten days (Hct 20) followed by adminstration of decitabine for ten days (0.52mg/kg/d; sc). RNA was isolated from nucleated erythroblasts purified from bone marrow aspirates obtained pre- and post-decitabine treatment. Purification of erythroblasts was performed by sedimentation in Percoll gradients followed by immunomagnetic column purification using an anti-baboon RBC antibody (Pharmingen). To assess the feasibility of using human Genechip arrays to detect differences in expression of baboon transcripts, RNA isolated from purified erythroblasts of a single baboon pre- and post-decitabine was hybridized in triplicate to human Genechip Focus arrays (Affymetrix) containing over 8500 genes. The expression of 48 genes was increased >2 fold in the post-decitabine treated sample compared to the pre-treatment sample. Among the more highly induced genes were HLA-A (3 fold), HLA-B (5.7 fold), exportin 4 (3.9 fold), and splicing factor 3b1 (3.7 fold). Reverse transcriptase PCR using human primer sets was performed to analyze the expression of these genes in pre- and post-decitabine treated bone marrow erythroblasts in independent samples from three additional baboons. Induction of HLA-A, exportin 4, and splicing factor 3b1 was confirmed in all three post-decitabine treated samples. The exportin 4 gene encodes a protein involved in nuclear export of the Smad3 protein. Activated TGF-β receptors phosphorylate Smad3 and induce its nuclear import to affect gene transcription. Following dephosphorylation of Smad3 in the nucleus, transport of the protein to the cytoplasm mediated by exportin 4 has been proposed to allow the propagation of multiple rounds of activation by activated TGF-β receptors thus amplifying TGF-β signaling (Kurisaki et al; Mol Cell Biol 26:1318, 2006). Because TGF-β increases HbF synthesis in cultured erythroid progenitors and also induces erythroid and megakaryocytic differentiation, we suggest that induction of exportin 4 by decitabine may play a role in the ability of this drug to increase HbF synthesis and alter hematopoietic differentiation. Our results thus confirm the feasibility of using human Genechip arrays to assess gene expression levels in baboons. Furthermore, we have indentified a gene induced by decitabine that potentially amplifies TGf-β signaling and thus may play a role in the ability of this drug to increase HbF and alter hematopoietic differentiation.

Comparative sensitivity of solid phase versus PEG enhancement assays for detection and identification of RBC antibodies

Blood banks require a sensitive, specific, and efficient method to detect clinically significant RBC antibodies. Solid phase antibody screening methods are popular due to high sensitivity and automation. However, the high degree of reactivity detects “false positive” antibodies of questionable clinical significance leading to additional testing. We studied positive rates of Capture-R vs. PEG methods and categorized RBC antibodies identified by initial test results of 33,564 consecutive samples by Capture-R method. Capture-R was positive in 1084/33,564 (3.2%) of samples. Using PEG as our “gold standard”, PEG confirmed true positivity (i.e., ⩾1 cell reacting) in 710 Capture-R positive samples (65.5%); 374 Capture-R positive samples (34.5%) did not react in PEG (i.e., false positives). Of the 710 samples with true positivity, only 510 showed clinically significant alloantibodies. Using PEG as our “gold standard”, only 2/3 of reactions by Capture-R were considered true positives. Because of ease and automation, Capture-R is popular as a screening test, but a more specific method may be helpful in order to identify truly significant alloantibodies.

Prevalence of HLA antibodies in transfused patients with and without red cell antibodies

Multiply transfused patients are at increased risk of developing red cell (RBC) antibodies, as well as antibodies to HLA. Although pretransfusion testing screens for RBC antibodies, no such testing is routinely performed for HLA antibodies. Determining which patients are more likely to make HLA antibodies may be important for patients undergoing elective surgery where platelets (PLTs) may be required. It is hypothesized that patients with RBC alloantibodies may be more likely to have HLA antibodies than previously transfused patients without RBC antibodies. Blood was collected from 53 adult male surgical patients with RBC alloantibodies and a control group of 69 similar male patients with a history of previous transfusions but no evidence of RBC alloimmunization. The samples were tested for the presence of immunoglobulin G Class I HLA antibodies by enzyme-linked immunosorbent assay. Of the 53 samples from patients with RBC alloantibodies, 12 (22.6%) also had HLA antibodies, whereas only 7 (10.1%) of the 69 patients in the control group had HLA antibodies (p < 0.03). There is a significant difference between the rates of HLA alloimmunization in male patients with RBC antibodies versus multiply transfused patients without RBC antibodies. Screening for HLA antibodies may be warranted in patients with RBC alloantibodies who might require PLT transfusion support for elective surgery.

In search of the Holy Grail: comparison of antibody screening methods

Currently, the goal of testing for RBC antibodies is to use a method that will detect, if possible, all antibodies that are considered clinically significant and yet not detect antibodies of little clinical importance in transfusion or pregnancy. The focus of test method development has been on the more controllable variables of the first and second stages of agglutination. Tube test methods have been developed over the years to achieve shorter turnaround times for quicker test results and improved sensitivity,with the occasional negative impact on relevant results. The focus on improving efficiency through automation, and personnel resourcing challenges of the transfusion service, have led laboratories to select methods tailored to meet their needs. This review compares the newer methods used in the gel test and solid phase test with commonly used tube methods. Both of the newer methods were developed with future adaptation to automation in mind. Further literature reviews about antibodies detected in only one or two methods and their general lack of clinical relevance as well as the occasional rare examples that produce significant clinical effects on transfused patients are also discussed.

Red blood cell alloantibodies after transfusion: factors influencing incidence and specificity

Alloimmunization after exposure to red cell (RBC) alloantigens depends on genetic and acquired patient-related factors, dose and route of administration, and the immunogenicity of the antigen, but exact kinetics are still unknown. A 5-year retrospective multicenter study analyzing factors influencing the rate and specificity of RBC alloimmunization was performed, with special emphasis on the time interval between transfusion event and antibody detection. Included were clinically significant alloantibodies against the Rhesus, Kell, Duffy, Kidd, and MSs blood group systems. Multivariate analysis involving 1710 immunized patients revealed that time interval between transfusion and antibody tests was strongly associated with the antibody specificity. Anti-Jk(a) and anti-Jk(b) were predominantly found in patients tested within 3 months, whereas anti-K and anti-Fy(a) were the most encountered antibodies at more than 5 years after transfusion. Of all immunized patients, new antibodies were detected within 14 days after transfusion in 299 patients (16.8%) and in 1479 patients (83.2%) after more than 14 days. Fifty percent of transfusion recipients were retested for alloimmunization because of a new transfusion indication. Eleven of 2932 patients (0.4%) retested up to 3 days after transfusion had formed new antibodies. The time interval between transfusion and antibody test was associated with RBC antibody specificity. Because RBC antibody tests after transfusion are not routinely performed, many antibodies may (not) be detected at the time of a new transfusion event, posing the transfusion recipient at risk for transfusion delay or a (delayed) hemolytic transfusion reaction. Routine RBC antibody screening at set time intervals after transfusion would reduce these risks.

Nonhemolytic antibody-induced loss of erythrocyte surface antigen

AbstractTransfusion of red blood cells (RBC150) into patients with anti–donor RBC antibodies (crossmatch-incompatible transfusion) can result in lethal antibody-mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival or adversely affecting the transfusion recipient. In an effort to elucidate the mechanistic details of this process, we describe the first animal model of nonhemolytic antibody-induced RBC antigen loss. RBCs from transgenic mHEL mice express surface hen egg lysozyme (HEL) as a transmembrane protein. Transfusion of mHEL RBCs into mice immunized with HEL results in selective loss of HEL antigen from donor RBCs without affecting other blood group antigens or reducing the circulatory life span of the transfused RBCs. While this process does not require the presence of a spleen, it requires both anti-RBC immunoglobulin G (IgG) antibodies and the FcγIII receptor. These studies provide mechanistic insight into the phenomenon of antigen loss during incompatible transfusion in humans.

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service

Case report: immune anti-D stimulated by transfusion of fresh frozen plasma

Abstract FFP has occasionally been reported to generate an immune response to RBC antigens (e.g., anti-D and anti-Fya). The Council of Europe requires that each unit of FFP have less than 6 × 109/L RBCs. However, there is considerable variation internationally in the method of production and the level and assessment of RBC contamination of FFP. This study reports the case of a 63-year-old group B, D– man who received multiple transfusions of D– blood products over a 4-month period. Seven months later the patient’s antibody screen remained negative and he was transfused with seven units of D– RBCs and six units of FFP, four of which were D+. Two months later anti-D, -E, and -K were detected in his plasma. Although the anti-E and anti-K could have resulted from transfusion of antigen-positive RBCs, the anti-D could have resulted only from transfusion of the D+ FFP. The D status of FFP is currently not considered when selecting products for transfusion even though the D antigen is highly immunogenic and the level of RBC contamination of FFP is not always known. This case highlights that transfusion of FFP is a stimulus for RBC antibodies and that when a patient has had a recent transfusion of FFP, consideration should be given to obtaining a sample for pretransfusion testing within 3 days before a scheduled RBC transfusion. In addition, the D status of FFP should be considered before administering FFP to premenopausal D– women. Immunohematology 2005;21:149–51.

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service

Evaluation of an Automated System for ABO/D Typing and RBC Antibody Detection System in a Hospital Transfusion Service

The Effect of Decitabine on Covalent Histone Modifications of Chromatin Associated with the ε-,γ -, and β-Globin Gene Promoters in the Baboon (P. anubis).

Treatment with the DNA demethylating drug decitabine increased HbF and F cells to therapeutically significant levels in patients with sickle cell disease in clinical trials. To gain more insight into the mechanism of action of this drug and to increase our understanding of the relationship between DNA methylation and chromatin structure we have determined the effect of decitabine on DNA methylation and covalent histone modifications of chromatin associated with the ε-, γ-, and β-globin gene promoters in purified erythroid bone marrow-derived cells obtained from 2 baboons (P. anubis) pre- and post-treatment. Baboons were phlebotomized for 10 days to maintain a hematocrit of 20 followed by subcutaneous decitabine administration (0.52mg/kg/day; 10–13 days). Erythroid cells (30–50 X 106) were purified from pre and post-treatment bone marrow apirates by immunomagnetic columns using an anti-baboon RBC antibody. The level of DNA methylation of 3 sites in the ε-globin promoter and 5 sites in the γ-globin promoter was determined by sequence analysis of ten cloned PCR products of each sample following bisulfite modification. Levels of ac-H3, ac-H4, dimethyl H3 ly4 (H3-dimeK4), dimethyl H3 ly79 (H3-meK79, dimethyl H3 ly36 (H3-meK36), and RNA pol II associated with the ε-, γ-, and β-globin promoters were determined by chromatin immunoprecipitation of sheared, formaldehyde fixed chromatin followed by real time PCR. In pre-treatment samples the level of DNA methylation of the γ-globin promoter (75%, 59.5%) was lower than the ε-globin promoter (84.%, 83.3%) and was associated with a small increase in HbF (5.4%, 9.0%). Following decitabine treatment HbF increased 8–10 fold (51.1%, 76%) and the level of DNA methylation of the ε-globin promoter (30.5%, 38.8%) and γ-globin promoter (24.3%, 25.0%) decreased 2–3 fold. Binding of RNA pol II to βthe -promoter was 2–3 fold higher than to the γ-promoter in pre-treatment samples. Following decitabine treatment the level of pol II associated with the γ-globin promoter was at least tenfold higher than with the β-globin promoter. In pre-treatment samples the level of ac-H3 and ac-H4 associated with the β-globin promoter was 2–4 fold higher than with the γ-globin promoter and 50 fold higher than with the ε-globin promoter. Following decitabine treatment the level of ac-H3 associated with the γ-promoter was 3–4 fold higher than with the β-promoter, while the level of ac-H4 associated with the γ-promoter increased to a level equivalent to that bound to the β-promoter. While no difference in the pattern of H3-dimeK4 associated with the ε-, γ-, and β-globin promoters was observed between pre- and post-treatment samples, minor changes in H3-meK79 and H3-meK36 were observed. These results demonstrate that decitabine treatment decreases DNA methylation of both the ε- and γ-globin promoters, greatly increases binding of RNA pol II to the γ-globin promoter, and increases the levels of ac-H3 and ac-H4 in chromatin associated with the γ-globin promoter. These experiments illustrate the usefulness of the baboon model to investigate the mechanism of pharmacologic reactivation of HbF synthesis at the molecular level.

Classical pathway complement destruction is not responsible for the loss of human erythrocytes during porcine liver perfusion.

Porcine livers perfused with human blood destroy 85% of human erythrocytes (red blood cells [RBC]) during prolonged extracorporeal perfusion, raising the possibility of a complement-mediated graft-versus-host effect. Isolated porcine livers were perfused with fresh human blood. Plasma samples were analyzed for complement production by reverse CH50 analysis and porcine immunoglobulin class and specificity by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Anti-CD59 and anti-decay accelerating factor (DAF) monoclonal antibody were used to investigate whether human complement regulatory proteins inhibit porcine complement. After 64 hr of perfusion of porcine livers with human blood, mean complement activity in the perfusate was 95% of the starting value and increasing, whereas perfusion in the absence of a liver showed a falling complement activity of 28.7%. ELISA demonstrated porcine immunoglobulin (Ig) G and IgM in the xenoperfused human plasma. Whereas in a previous study flow cytometry demonstrated porcine antibodies specific for antigens on human T lymphocytes, in this study, anti-human RBC antibodies were not found. Xenoperfused human plasma did not lyse fresh human RBC. Human complement was consistently more efficient at lysing porcine RBC than was porcine complement at lysing human RBC, and human plasma inhibited the ability of porcine plasma to lyse human RBC, raising the possibility of cross-species complement regulation. Complement regulatory proteins on human RBC were blocked using mouse monoclonal anti-human CD59 and DAF. Blocking CD59, but not DAF, augmented lysis of human RBC by porcine complement. Human CD59 inhibits porcine complement. The production of porcine complement from xenoperfused porcine livers is unlikely to result in clinically significant injury mediated through the classical pathway of complement activation.

The importance of antibodies against low-incidence RBC antigens in complete and abbreviated cross-matching.

It is common practice to perform an antiglobulin cross-match only when unexpected RBC alloantibodies are present, to detect antibodies against additional RBC antigens. In this study, the incidence of unexpected antibodies to low-incidence antigens (Ab-LIA) over a period of 23 years was investigated. Records of RBC antibodies and the accompanying transfusion history from 1978 through 2000 was retrospectively examined. Complete cross-matches were performed for all RBC transfusions before 1991. As of 1991, the type-and-screen policy was applied. To study the incidence of anti-Wra, a prospective study was conducted on sera from 462 patients sent to the transfusion laboratory and 486 blood donors. The records of 1795 patients containing 2257 RBC antibodies were examined. In 89 patients, a total of 94 Ab-LIAs was found. Anti-Wra was the most frequently encountered Ab-LIA. Thirty-nine patients had Ab-LIA in combination with other antibodies, 20 of which were autoantibodies. Eighty percent of these Ab-LIA were found at the first positive antibody screening test. Fifty-one solitary Ab-LIA were found in 50 patients, 37 during antibody screening tests, and 14 after positive complete cross-matches conducted before 1991. After an RBC antibody was detected, 664 patients received a total of 7792 RBC transfusions. Since the introduction of the type-and-screen policy, only one anti-Wra has been discovered during complete cross-matching. No transfusion reactions due to Ab-LIA were reported during the study period. In the prospective study, 12.3 percent of patients and 4.3 percent of blood donors had anti-Wra. Although Ab-LIAs are found coincidentally in the sera of only 2 to 3 percent of patients with other RBC antibodies, they are formed often. Because we found no difference in serologic incompatibility, due to Ab-LIAs, between patients with and without other blood group antibodies, we conclude that blood can be transfused safely to patients without performing a complete cross-match.

Probability of anti-D development in D- patients receiving D+ RBCs.

In some situations, the administration of D+ RBCs to D- patients is necessary. The probability of a subsequent anti-D formation is assumed to be around 80 percent, a figure based primarily on studies in healthy volunteers. It was hypothesized that patients requiring blood transfusion have a much lower probability of developing antibodies. A retrospective analysis was performed whereby 78 D- patients were evaluated for the development of RBC antibodies after administration of D+ RBCs. For the analysis of the cross-sectional observations, parametric models were used for interval-censored data. Anti-D was detected in 16 of 78 patients. Considering the individual patient's inspection times, the calculated probability of developing antibody following D+ RBC supply was shown to be below 41.7 percent (upper 95% confidence bound) and estimated as 30.4 percent. The data hinted toward an inverse correlation between the number of transfused units and the probability of antibody formation. Interestingly, 6 of these 16 patients developed additional IgG autoantibody. In 3 of those cases, evidence for prolonged hemolysis was found. The actual frequency of antibody formation in our patients is much lower than assumed. On the other hand, prolonged hemolysis probably induced by additional autoreactive antibodies might occur. This possible complication has not yet been addressed. Further studies might reveal whether a less restricted transfusion policy with respect to D matching is justified in selected patients.

Letter to the Editors: Irregular RBC antibodies in the sera of Brazilian pregnant women

Letter to the Editors: Irregular RBC antibodies in the sera of Brazilian pregnant women

Antibodies detected in samples from 21,730 pregnant women

Abstract Although anti-D is still the main cause of HDN, many other antibodies have been implicated. From September 1995 to April 2000, screening for RBC antibodies was performed on samples from 21,730 pregnant women regardless of RhD type. Standard tube and gel methods were used. Anti-D was identified in 254 samples; other antibody specificities were detected in 376 samples, for a total of 630 antibodies. For this study, 522 antibodies were considered clinically significant. The incidence of potentially clinically significant antibodies was 2.4 percent. The majority belonged to the Rh system, followed by anti-M, -Fya, -S, -Jka, and -Jkb. Among antibodies of no clinical significance, the most frequent were antiH, -Lea, and -P1.

Expression, purification, and characterization of an anti-RBCFab-p24 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood

A fusion protein A41Fabp246 consisting of Fab of an anti-RBC MAb A41 and amino acids 8-153 of HIV-1 p24 (p246) fused at the C-terminus of A41Fd was purified following assembly of A41Fdp246 with A41 LC in vitro, using a denaturation– renaturation protocol and a 4-step column chromatography procedure. The highly purified, monomeric A41Fabp246 was then evaluated for hemagglutination-based detection of anti-p24 antibodies using sera from HIV-infected individuals. This derivative of p24 is devoid of maximum homology region and the C-terminal domain of p24, which is responsible for oligomerization of p24, but retains full complement of immunodominant epitopes. This new fusion protein in combination with fusion proteins consisting of monovalent fragment of another anti-human RBC antibody fused to immunodominant regions of envelope glycoproteins of HIV-1 and HIV-2 should be useful in preparing a cocktail of reagents for highly sensitive detection of anti-HIV antibodies in whole blood.

RBC alloantibody specificity and antigen potency in Olmsted County, Minnesota.

While RBC antigen frequencies for whites of Northern European ancestry are known, the relative frequencies of RBC antibodies within this population have not been determined. The distribution of RBC alloantibodies by sex and age was studied, as were the immunogenicity of RBC antigens and the occurrence of RBC alloantibody clusters in a geographically defined population. RBC alloimmunization among patients and donors in Olmsted County, MN, was determined for the period from 1975 to 1995. Alloantibody frequencies were used to calculate the potency of each antigen relative to K. Cluster analysis was applied to the data to identify natural groupings of antibodies. The frequency and potency of 33 alloantibodies from 1345 alloimmunized subjects were estimated. The most frequent alloantibodies were E (20.8%), Le(a) (18.6%), K (14.7%), D (12.9%), Le(b) (9.4%), M (7.2%), P(1) (6.7%), Fy(a) (6.3%), C (6.8%), and c (3.5%). The most potent antigens were Wr(a) (0.363), C(w) (0.078), Le(a) (0.03), E (0.028), V (0.025), Js(a) (0.023), Kp(b) (0.023), Go(a) (0.023), JMH (0.023), and Rd (0.023). Greater frequency of overall alloimmunization (M:F = 1:2.7), anti-D (p<0.0001), and anti-Le(a) (p = 0.003) was seen among females. Warm autoantibodies were more frequent among males with positive antibody screens (p<0.0001). No other gender differences were observed. Alloimmunization increased with age for K, Kp(a), Fy(a), D, C, E, and warm autoantibodies. Frequencies of alloimmunization to Le(a), Le(b), M, and P(1) decreased with age. The cluster analysis showed grouping of the antibodies to C and D as well as to Le(a) and Le(b), but the other RBC alloantibodies did not form clusters. Less than 1 percent of residents tested had positive antibody screens. Anti-E and anti-Le(a) were more common than anti-K. Wr(a) and C(w) were more potent antigens than K. Most antibodies showed an increase in frequency with increasing age. Except for anti-C and -D and anti-Le(a) and -Le(b), RBC alloantibodies did not occur in clusters.