Modeling alloantibody formation to high-incidence red blood cell antigens in immune responders using genotypic data.

CONCLUSIONS: Alloimmunization to red blood cell antigens is unpredictable and poorly understood. Patients who are negative for high-incidence antigens (HIAs) are at risk for developing the corresponding antibodies. Molecular methods can easily predict the lack of an antigen and thus, the risk of an individual to become immunized. We examined the prevalence and risk factors for HIA alloimmunization in patients at risk based on genotyping results. Genotyping using a molecular method (HEA BeadChip™, Immucor, Warren, NJ) was performed on all patient specimens referred for molecular testing over 45 months; serologic and clinical data were analyzed. We used simple and multiple logistic regression to model the risk factors for alloimmunization to an HIA. Of the 2591 patients genotyped, 32 (1.2%) were homozygous for at least one variant predicting absence of an HIA. Of these 32 patients, prior transfusion or pregnancy history was available for 29 (91%). Four susceptible patients made an antibody to an HIA (12.5% of all, 13.8% of those with a documented exposure). Two of these four patients (50%) had made an alloantibody to another antigen. The odds of forming an antibody to an HIA were not related to the total number of transfusions (p = 0.47), the total number of alloantibodies (p = 0.61), or diagnosis of sickle cell disease (p = 0.77) in simple logistic regression. Adjustment for the other two variables in a multiple logistic regression was also not significant for each variable (p = 0.6, p = 0.7, and p = 0.7, respectively). Although they had a known exposure to alloantigens through transfusion or pregnancy, 86.2 percent of patients (25 of 29) at risk for alloantibody formation to an HIA in fact did not mount an immune response to that antigen. Possible risk factors including the number of transfused units or the total number of alloantibodies made were not predictors of making an alloantibody to an HIA in our sampling. Our results suggest that other patient-specific risk factors for alloimmunization exist.

ABO antibody titer and risk of antibody-mediated rejection in ABO-incompatible renal transplantation.

Therapeutic plasma exchange (TPE) preconditioning with immunosuppressive therapy reduces ABO antibody titers, permitting engraftment of ABO-incompatible (ABO-I) kidney transplants. The posttransplant predictive role of ABO antibody titers for antibody-mediated rejection (AMR) is unknown. This retrospective study evaluated 46 individuals who received TPE to permit ABO-I kidney transplantation. ABO antibody titers were performed using donor-type indicator red cells. Seven individuals (15.2%) experienced clinical or subclinical AMR. There was no significant difference between recipient blood group, number of pretransplant TPE and baseline titer between those with and without AMR. At 1-2 weeks posttransplant the median titer was 64 (range 4 - 512) among individuals with AMR and 16 (range 2 - 256) among individuals without AMR. Total agglutination reactivity score was significantly higher among individuals with AMR (p = 0.046). The risk of AMR was significantly higher among individuals with an elevated posttransplant titer of >or=64 (p = 0.006). The sensitivity of an elevated posttransplant titer was 57.1% with a specificity of 79.5%. The positive predictive value was 33.3% and the negative predictive value was 91.2%. Most individuals with AMR have an elevated titer, however, the positive predictive value of a high titer for AMR is poor.

Prophylactic antigen-matched donor blood for patients with warm autoantibodies: an algorithm for transfusion management.

BACKGROUND: Patients with warm autoantibodies are at high risk for delayed hemolytic transfusion reactions due to the presence of alloantibodies. To provide blood safe for transfusion and to avoid adsorption studies in some cases, the provision of prophylactic antigen-matched donor blood where feasible for patients with warm autoantibodies is advocated. STUDY DESIGN AND METHODS: Twenty consecutive adult patients with warm autoantibodies (January 1999 to February 2000) received chronic RBC transfusions by use of this protocol: the serology consistent with warm autoantibodies was confirmed; the alloantibodies were identified; the complete phenotype was determined (i.e., C, E, c, e, K, Jk(a), Jk(b), Fy(a), Fy(b), S, and s); and prophylactic antigen-matched (i.e., donor RBCs matched with the patient's phenotype), WBC-reduced donor RBCs were provided for transfusion. On subsequent admissions, samples were evaluated by panel studies and DATs. If the serology remained consistent with previous findings, prophylactic antigen-matched, WBC-reduced RBCs were transfused without further testing. RESULTS: Eight of 20 (40%) patients had existing, clinically significant alloantibodies. In 12 of 20 (60%) patients, a phenotype was determined and the patients received transfusion of a total of 149 prophylactic antigen-matched RBC units (mean, 15 units per patient) precluding adsorption studies on 51 pretransfusion samples. In 8 of 20 (40%) cases (2 with alloantibodies), phenotypes were indeterminant, necessitating differential allogeneic adsorption studies on 39 samples before transfusion of 144 RBC units (mean, 18 units per patient). CONCLUSIONS: Determining complete phenotypes should be a routine component of the serologic evaluation of patients with warm autoantibodies. Our algorithm for providing prophylactic antigen-matched RBCs to these patients when a complete phenotype can be determined provides flexibility in their transfusion management while maintaining safety and circumvents or simplifies pretransfusion adsorption studies.

An animal model for delayed hemolytic transfusion reactions.

Delayed hemolytic transfusion reactions (DHTRs) are a well-known complication of transfusion that may be defined as immune-mediated hemolysis of allogeneic donor red cells that occurs approximately 3 to 5 days after transfusion. In general, DHTRs occur in patients who have been alloimmunized previously, but the antibody titers have fallen below serologically detectable levels. Transfusion of seemingly compatible blood and exposure to the putative alloantigen results in an anamnestic immune response that may lead to in vivo accelerated destruction of donor red cells. Symptoms may include a drop in hemoglobin and hematocrit, fever, jaundice, and renal insufficiency. More recent studies have shown that there is a subset of cases called delayed serologic transfusion reactions (DSTRs) when there are serologic findings consistent with DHTRs but no clinical evidence of hemolysis. In both DHTRs and DSTRs, direct antiglobulin tests are often persistently positive long after the transfused donor red cells should have been removed from the circulation. Because the studies required to investigate the immunologic and clinical aspects of these reactions are precluded in humans, we developed an animal model for the study of DHTRs and DSTRs. Our article provides a comprehensive review of DHTRs and DSTRs, the role of complement and cytokines in these reactions, and the phenomenon of bystander hemolysis. We describe our studies using the rabbit as a model for the study of DHTRs and bystander hemolysis.

Fatal warm autoimmune hemolytic anemia resulting from IgM autoagglutinins in an infant with severe combined immunodeficiency.

Autoimmune diseases are rare in patients with severe combined immunodeficiency (SCID). The authors describe an 11-month-old infant girl with SCID with fatal warm autoimmune hemolytic anemia (AIHA) resulting from IgM autoagglutinins. Serologic evaluation revealed IgM autoantibodies that caused in vitro hemagglutination at 37 degrees C. The patient had clinical evidence of ongoing hemolysis and agglutination despite aggressive treatment. She had three strokes and died 6 weeks after unsuccessful bone marrow transplantation. Autoimmune disease is an unexpected complication of SCID. The presence of warm reactive IgM autoagglutinins in AIHA confers a dismal prognosis.

An acute hemolytic transfusion reaction due to anti-IH in a patient with sickle cell disease.

BACKGROUND: A hemolytic transfusion reaction (HTR) due to anti-IH is reported in a patient with sickle cell disease (SCD). CASE REPORT: An 18-year-old woman with SCD and a complete phenotype on file had been identified as group B-positive with negative antibody-screening tests and had received 1 unit of packed RBCs. Ten days later, she was readmitted in painful crisis with a Hb of 4.2 g per dL. Antibody-screening tests and panel cells were positive at all test phases with a negative autocontrol, which suggested alloantibodies. Phenotypically matched group O RBCs were issued emergently. After the transfusion of 100 mL, the patient had an HTR with chills, fever, and tachycardia and laboratory findings of hemoglobinemia, hemoglobinuria, and negative DATs. A high-titer, IgM anti-IH with a high thermal amplitude (reactive with group O, but not group B RBCs at 37 degrees C) was identified. Autologous RBCs appeared to have normal I antigen expression, but less H antigen than pooled group B RBCs. She was given group B RBCs, uneventfully, by use of a blood warmer. CONCLUSIONS: This is a rare case of anti-IH as the cause of a HTR, as a serologic problem that may be seen in SCD, and as an autoantibody that may mimic an alloantibody. Ironically, this HTR resulted from the effort to provide phenotypically matched RBCs, which necessitated the selection of group O RBCs.

A comparison of a new affinity column system with a conventional tube LISS-antiglobulin test for antibody detection.

A recently introduced system for antibody detection (ReACT) consists of affinity columns (AFC) that contain protein A and protein G-coated agarose. We compared the ReACT system to a conventional tube low-ionic-strength saline antiglobulin test (LISS-AGT). We selected 100 LISS-AGT positive samples with clinically important and benign antibodies of varying strengths and 130 LISS-AGT negative samples to evaluate by the AFC method. AFC tests were positive with all 84 clinically important antibodies, including 36 antibodies that reacted <= 1+ at LISS-AGT (0% falsely negative). Eleven of 16 (69%) clinically benign antibodies reacted by AFC. Five samples (2 anti-Sda, 2 anti-I, and 1 inconclusive) were negative by AFC. AFC tests were negative with all 130 samples that were negative by LISS-AGT (0% falsely positive). The AFC method showed results comparable with results obtained with a conventional tube LISS-AGT for detection of clinically important antibodies. Some unwanted, clinically benign antibodies may not be detected by the AFC method.

Assessment of the clinical significance of anti-Dob.

BACKGROUND: Anti-Dob is an uncommon antibody, and there are few data regarding its clinical importance. In the present case, the patient's transfusion management was based on both in vivo and in vitro assay results. CASE REPORT: A delayed hemolytic transfusion reaction was suspected in a 64-year-old white woman awaiting cardiac surgery when the transfusion of 1 unit of red cells failed to raise her hematocrit. Although direct antiglobulin tests were negative, antibody screening tests on samples drawn 9 days after transfusion were positive, and anti-Dob was identified, reacting to a titer of 4. 51Cr in vivo survival studies with incompatible Do(b+) red cells showed poor survival: 83.2 percent at 1 hour, 43 percent at 24 hours, and 29.6 percent at 48 hours and t1/2 = 19 hours (normal t1/2 = 25-35 days). A monocyte monolayer assay performed with the same incompatible Do(b+) donor red cells also indicated poor survival: 22 percent and 30 percent reactive monocytes, respectively, with and without the addition of complement (normal, 0-3%). The patient was given 4 Do(b-) red cell units without clinical signs or symptoms of a reaction. CONCLUSIONS: This example of anti-Dob was implicated in a delayed hemolytic transfusion reaction. The 51Cr survival studies and monocyte monolayer assay results indicated that the anti-Dob was clinically significant, requiring the use of Do(b-) red cells for transfusion.

Fatal autoimmune hemolytic anemia in a child due to warm-reactive immunoglobulin M antibody.

PURPOSE: Autoimmune hemolytic anemia (AIHA) due to warm-reactive immunoglobulin M (IgM) antibodies is rare in adults and has never been described in children. This report describes a pediatric patient with warm AIHA due to high-titer complete IgM antibody. PATIENTS AND METHODS: A 9-year-old girl with a history of Evan's syndrome had severe anemia, fatigue, and skin mottling. RESULTS: Serologic evaluation revealed a high-titer, high thermal amplitude (37 degrees C) complete IgM autoantibody. Despite aggressive management (including high dose corticosteroids, intravenous immune globulin, cyclophosphamide, mycophenolate mofetil, whole blood exchange transfusions, and cyclosporine A), the patient remained markedly anemic and developed multiorgan system failure related to diffuse in vivo hemagglutination. Her clinical course included cardiovascular collapse caused by agglutinated red blood cells in the right ventricle with outflow obstruction, cerebrovascular infarcts, hepatic failure, and infarction of her extremities. She ultimately died from disseminated Aspergillosis infection. CONCLUSION: This rare form of AIHA is associated with a dismal prognosis. Early, aggressive treatment is advocated, although it remains to be seen whether the clinical course can be reversed and the outcome improved.

Differentiation of anti-D, -C, and -G: clinical relevance in alloimmunized pregnancies.

BACKGROUND: The differentiation of anti-D, -C, and -G specificities is seldom considered clinically important in pretransfusion testing. However, distinguishing these antibody specificities in alloimmunized pregnancies may be essential. The clinical prognosis as well as Rh immune globulin prophylaxis depends on the accurate identification of these antibodies. CASE REPORT: A pregnant woman, para 1 gravida 4, who had received Rh immune globulin at appropriate intervals during her previous pregnancies was reported to have anti-D (titer = 4) and anti-C (titer = 32). Differential adsorption and elution studies showed that the patient had anti-C and anti-G, but not anti-D. This case prompted retrospective examination of the sera from six other women with anti-D and anti-C who were referred to a high-risk pregnancy clinic. Of six pregnant women reported to have anti-D and anti-C; two had anti-D, -C, and -G; three had anti-D and -G, but not anti-C; and one had anti-C and -G, but not anti-D. This last is similar to the index case. CONCLUSION: Cases of pregnant women with anti-C and -G, but not anti-D, are not infrequent. Studies to differentiate anti-D, -C, and -G should be performed on alloimmunized pregnant women presumptively identified as having anti-D and anti-C when the medical history (Rh immune globulin prophylactic therapy) and/or titer values (e.g., anti-C titer higher than anti-D titer) suggest that anti-D may not actually be present. Rh immune globulin has not failed in these patients, and they should receive this therapy during pregnancy to prevent immunization to D.

Delayed hemolytic transfusion reactions in sickle cell disease: simultaneous destruction of recipients' red cells.

BACKGROUND: Bystander hemolysis may be defined as the destruction of antigen-negative red cells during immune hemolysis, such as delayed hemolytic transfusion reaction (DHTR). Although many have suspected that bystander hemolysis does occur, that phenomenon is very difficult to document. STUDY DESIGN AND METHODS: Five patients with sickle cell disease (SCD) who underwent exchange transfusion and subsequently experienced a DHTR were retrospectively evaluated. Serial samples were examined for complete blood counts, the percentage of hemoglobin A and S, and the percentage of reticulocytes. The total red cell count and the percentage of hemoglobin S were used to calculate the hemoglobin S red cell count. The patients' profiles were compared to proposed models. RESULTS: DHTRs due to anti-E, -S, -Fy(a), or -Jk(a) or serologically undetectable antibodies were identified 7 to 19 days after exchange transfusion. All patients had a significant decrease in hemoglobin A red cells; 56.4 to 94.7 percent of hemoglobin A red cells were hemolyzed. Patients 4 and 5 had a decrease in hemoglobin S red cells which indicated the destruction of autologous red cells during DHTR. The evidence for bystander hemolysis was particularly convincing in Patient 5, because there was a substantial decrease in hemoglobin S red cells despite a reticulocyte production index of 2.2 percent at the nadir of the DHTR. CONCLUSION: Hemoglobin S provides a biologic marker for monitoring autologous red cell loss in sickle cell patients. We have shown one patient with clinical evidence of bystander hemolysis complicating a DHTR.

Hemolysis due to the simultaneous occurrence of passenger lymphocyte syndrome and a delayed hemolytic transfusion reaction in a liver transplant patient.

Hemolysis due to donor-derived antibodies produced by "passenger" B lymphocytes, called passenger lymphocyte syndrome, has been described in ABO-unmatched solid organ and bone marrow transplant recipients. Delayed hemolytic transfusion reactions occur within a similar time frame and have similar clinical and serologic findings. To our knowledge, we report the first case of hemolysis due to the simultaneous occurrence of passenger lymphocyte syndrome (donor-derived anti-A) and a delayed hemolytic transfusion reaction (recipient-produced anti-E) in a liver transplant patient.

Tacrolimus-related posttransplant lymphoproliferative disorder presenting as autoimmune hemolytic anemia.

Tacrolimus (formerly known as FK506) is a macrolide immunosuppressant that has been used to prevent rejection of solid organ allografts. Acute hemolytic anemia is one of the side effects associated with tacrolimus therapy, and two mechanisms have been described to account for acute hemolytic anemia in patients receiving tacrolimus: drug-induced hemolysis and alloimmune hemolysis resulting from donor lymphocytes derived from the allograft (passenger lymphocyte syndrome). We report a case of a liver transplant recipient who developed fatal autoimmune hemolytic anemia while under treatment with tacrolimus for allograft rejection, and in whom postmortem examination revealed a clinically unsuspected posttransplant lymphoproliferative disorder. This case implicates autoimmune hemolytic anemia as a novel mechanism of acute hemolysis in patients treated with tacrolimus and further suggests that acute hemolytic anemia in this group of patients may herald an occult lymphoproliferative disorder.

Leukocyte reduction of red cells when transfusing patients with autoimmune hemolytic anemia: a strategy to decrease the incidence of confounding transfusion reactions.

Autoimmune hemolytic anemia (AIHA) presents a difficult challenge to clinicians and blood bankers alike. Autoantibodies in the serum significantly complicate serologic evaluation, and necessitate performing procedures such as adsorptions to eliminate the possibility of underlying alloantibodies. In many instances the blood that is issued may be phenotypically similar but remains crossmatch incompatible, generating a considerable degree of anxiety among the clinical staff who are responsible for transfusing the patient. We report a case of warm autoimmune hemolytic anemia (WAIHA) in which the transfusion of red cells was complicated by a febrile transfusion reaction. Evaluation of the reaction resulted in a significant delay in transfusion therapy. Subsequent administration of leukocyte-poor red cells resulted in uneventful transfusions with a good therapeutic response. Retrospective analysis of the pretransfusion sample demonstrated significant levels of anti-neutrophil antibodies. This case resulted in the establishment of our policy to administer all red cell transfusions to patients with autoantibodies (warm or cold) as leukocyte- poor red cells.

A quantitative assay for subclassing IgG alloantibodies implicated in hemolytic disease of the newborn.

Traditionally, IgG subclassing has been performed using qualitative assays. Quantitation of IgG subclasses may have prognostic value in evaluating alloimmunized pregnancies. A quantitative enzyme-linked immunosorbent assay (ELISA) was implemented for measuring IgG subclasses of red blood cell (RBC) antibodies (AB) isolated by adsorption/elution from the sera of alloimmunized pregnant women. The assay is a sandwich enzyme immunoassay using monoclonal antibodies specific for the relevant IgG subclasses and anti-human IgG peroxidase conjugate to quantitate the amount of bound IgG. The sensitivities of the assay for IgG1, 2, 3, and 4, respectively, were 4, 23, 4 and 2 micrograms/l. The results for each subclass for a given AB were expressed as a percentage of the total. In a series of pregnant mothers with ABs: E (4), Fya (2), Jka (1) and S (1), the mean percentage +/- 1SD of each subclass was: IgG1 61 +/- 34; IgG2 14 +/- 22; IgG3 18 +/- 28 and IgG4 4 +/- 17. IgG1 or IgG3 accounted for greater than 50% of the AB subclass distribution in 5 cases that resulted in hemolytic disease of the newborn (HDN). Although only a small number of samples was studied, changes in the concentrations of IgG1 or IgG3 during gestation suggest a correlation with the presence or absence of HDN. The ELISA may be used to quantitate the IgG subclasses of RBC ABs and may be valuable in predicting the severity of HDN in alloimmunized pregnancies.

The risk of alloimmunization to c (Rh4) in R1R1 patients who present with anti-E.

BACKGROUND: Because the Rh antigens E (Rh3) and c (Rh4) are relatively immunogenic, it has been suggested that R1R1 (E-, c-) patients who present with anti-E alone receive prophylactic c- (Rh: -4) red cell transfusions. STUDY DESIGN AND METHODS: To determine the utility of this approach, the transfusion records of 100 consecutive R1R1 patients with anti-E identified over a 6-year period were reviewed. RESULTS: Thirty-two (32%) had anti-c concurrent with anti-E. Twenty-seven of the 68 patients who presented with anti-E alone received random (i.e., not typed for c [Rh4]) red cell transfusions. Five (18.5%) of the 27 subsequently developed anti-c 13 to 193 days (mean, 50) after transfusion of 2 to 14 (mean, 8) red cell units. None of the five had clinical evidence of hemolysis that could be attributed to a delayed hemolytic transfusion reaction. Twenty-two (81.5%) of the 27 failed to develop anti-c even after transfusion of 1 to 41 (mean, 9; median, 7) red cell units. CONCLUSION: The overall rate of immunization to c (Rh4) antigen in R1R1 patients with anti-E was 37 percent. Production of anti-c following transfusion to R1R1 patients with anti-E occurred in 18.5 percent of the cases in this series, which could have been avoided by the prophylactic use of R1R1 (E-, c-) blood for transfusion. The prophylactic use of c- (Rh: -4) blood in this patient population may be justified by the high immunization rate and the potential risk of delayed hemolytic transfusion reaction.

Association of Lewis blood group phenotypes with urinary tract infection in children.

Many blood group antigens, genetically controlled carbohydrate molecules, are found on the surface of uroepithelial cells and may affect bacterial adherence and increase the frequency of urinary tract infection (UTI) in adults. Sixty-two children aged 2 weeks to 17 years (mean, 2.3 years) who were hospitalized with fever in association with UTIs caused by Escherichia coli had complete (n = 50) or partial (n = 12) erythrocyte antigen typing to determine the role of erythrocyte antigens and phenotypes in UTI in children; 62 healthy children undergoing nonurologic elective surgery, matched 1 to 1 for age, sex, and race to the patient group, formed the control group. In univariate tests, patients and control subjects did not differ in ABO, Rh, P, Kell, Duffy, MNSs, and Kidd systems by the McNemar test of symmetry (p > 0.05). The frequency of the Lewis (Le) (a-b-) phenotype was higher (16/50 vs 5/50; p = 0.0076) and the frequency of the Le(a + b +) phenotype was lower (8/50 vs 16/50; p = 0.0455) in the patient population than in the control subjects. A stepwise logistic regression model to predict UTI with the explanatory variables A, B, O, M, N, S, s, Pl, Lea, and Leb showed that only the Lea and Leb antigens entered the model with p < 0.1. The Le(a-b-) phenotype was associated with UTI in this pediatric population. The relative risk of UTI in children with the Le(a-b-) phenotype was 3.2 (95% confidence interval, 1.3 to 7.9). Specific blood group phenotypes in pediatric populations may provide a means to identify children at risk of having UTI.

Polyethylene glycol versus low-ionic-strength solution in pretransfusion testing: a blinded comparison study.

BACKGROUND: Polyethylene glycol (PEG) has been shown to potentiate antigen-antibody reactions. STUDY DESIGN AND METHODS: To investigate the utility of PEG in pretransfusion testing, a blinded comparison study of PEG and a low-ionic-strength additive solution (LISS) was conducted. A total of 500 patient samples were tested in parallel with reagent antibody-detection cells using blind-coded PEG and LISS potentiators. RESULTS: In 34 (34%) of 100 samples with known antibodies in the Rh, Kell, Duffy, Kidd, and MNS systems, PEG antiglobulin reactions were stronger (total score, 382) than LISS antiglobulin reactions (total score, 216), and in 66 cases (66%), they were equal to those of LISS. Of 400 samples without detectable antibodies, 384 were negative with PEG and LISS, and 16 were positive in PEG tests and negative in LISS. Seven of the 16 were clinically important antibodies (D, 1; E, 3; Fya, 1; Jka; 1; Jkb, 1), and four were clinically benign antibodies (Le(a), 2; McCc, 1; Sda, 1). Five of the 16 demonstrated inconclusive PEG reactions, for a false-positive rate of 5 in 400 (1.3%). Of the 500 samples, none was negative in PEG tests and positive in LISS (0% false-negative rate). CONCLUSION: Although PEG demonstrates a relatively high false-positive rate, PEG is more sensitive than LISS in detecting clinically significant antibodies.

Compatibility testing problems associated with bovine thrombin-treated plasma.

Plasma converted to serum by the addition of bovine thrombin prior to compatibility testing agglutinated all donor red cells. This finding prompted an investigation of bovine thrombin- associated incompatibility that showed that (1) thrombin derived from bovine plasma contains IgG antibodies directed against all human red cells, and (2) excess (> 50 units/mL) bovine thrombin used for conversion of plasma to serum may cause hemagglutination and erroneous serologic test results because of the presence of heteroagglutinins.