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.

Fenoprofen-induced immune hemolysis. Difficulties in diagnosis and complications in compatibility testing.

A 70-year-old man developed severe immune intravascular hemolysis and renal failure following ingestion of fenoprofen, a nonsteroidal, anti-inflammatory drug. The patient's red blood cells were sensitized with both IgG and C3d. The serum reacted with normal red blood cells in the presence and absence of the drug. Addition of albumin to the serum inhibited the reactivity with both neat and drug-treated serum. These atypical serologic findings for drug-related immune hemolytic anemia were explained by (1) the measurement of fenoprofen by high performance liquid chromatography (HPLC) in the neat serum; and (2) solid-phase adsorption studies showing that albumin can bind drug, leading to the disappearance of agglutination when albumin is added. This case demonstrates the utility of drug levels and adsorption techniques to confirm the diagnosis of drug-induced immune hemolytic anemia despite the anomalous serologic results which obscured the diagnosis and management of the patient.

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.

Plasmapheresis for the treatment of repeated early pregnancy wastage associated with anti-P.

It has been proposed that the blood group antibody, anti-P, produced by p or Pk individuals may cause abortion early in pregnancy. The authors have studied and successfully treated a Pk woman with anti-P who had 13 consecutive first-trimester miscarriages. Anti-P was implicated as the cause of repeated pregnancy loss after extensive clinical, endocrinologic, immunologic, and chromosomal evaluations. To remove P blood group antibodies, plasmapheresis was begun at five weeks' gestation during the 14th pregnancy with one plasma volume exchange two to three times per week. This therapy resulted in a reduction in the titer of anti-P, and the patient was delivered of a viable female infant after 33 weeks' gestation. The management and outcome indicate that habitual abortion presumably due to anti-P can be successfully treated with plasmapheresis. This case provides additional evidence that anti-P is responsible for abortions in p or Pk women, and that these abortions are immunologically mediated.

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.

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.

Immune hemolytic anemia following heart-lung transplantation.

Immune hemolytic anemia due to minor ABO incompatibility between recipient and donor is a well-recognized occurrence in kidney and liver transplantation. In some cases, the responsible antibodies have been shown to be derived from the donor passenger lymphocytes using Gm allotyping. We report a case of acute, transient hemolysis following heart-lung transplantation in which serologic and Gm allotype studies confirmed the etiology of hemolysis.

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 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.

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.

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.

The Du phenotype, serology, and clinical relevance.

In this article, the authors examine the nature of the Du phenomenon through a comprehensive historical review beginning with the initial description of the Du factor in the 1940s. Pertinent developments in serologic testing methods and genetic concepts are described. Evidence of the importance of the Du factor in transfusion and hemolytic disease of the newborn is also presented. Selected articles on the frequency of Du in Caucasian and Negro populations are cited. Finally, the authors review current theoretical concepts concerning the nature of the Du factor, its importance in current transfusion practice and maternal Rh immune globulin administration, and the use of microscopic Du testing as a screening procedure for fetomaternal hemorrhage.

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.

The efficacy of performing red cell elution studies in the pretransfusion testing of patients with positive direct antiglobulin tests.

In order to evaluate the efficacy of performing red cell elutions in pretransfusion testing, the serologic records of 638 patients with positive direct antiglobulin tests (DAT) were reviewed. These patients were identified by routine antibody screening procedures that included an autologous control. DAT results on the red cells of these patients showed 279 with IgG and C3d sensitization, 319 with IgG alone, and 40 with C3d sensitization alone. Of 638 patients' red cell eluates, 401 demonstrated no reactivity, 154 demonstrated panagglutination, and 60 demonstrated passively acquired anti-A,B. Only 23 of 638 patients had alloantibody sensitization of their red cells. Of the 23, 19 had serum antibody corresponding to the specificity of antibody detected in the eluate. Thus, only four of 638 (0.6%) eluates gave results unavailable by serum testing alone. This study indicates that routine eluate investigation provides little useful information in assuring compatibility. Serum antibody testing and careful review of the clinical and transfusion history constitute appropriate pretransfusion testing in patients with positive direct antiglobulin tests. Eluate testing should be restricted to cases in which immune hemolysis is suspected clinically.

Probenecid induced immune hemolytic anemia.

We report a patient who, while receiving probenecid and colchicine for acute gouty arthritis, developed severe hemolytic anemia in association with a generalized rash. The hemolysis was immune mediated as shown by a positive direct Coombs' test. In vitro hematologic studies showed that a probenecid dependent antibody was present.

Immunohematologic complications of ABO-unmatched liver transplants.

Transplantation of ABO-unmatched livers has been associated with the development of donor-derived antibody (DDAb) and hemolysis. Nine (22%) of 41 consecutive patients undergoing liver transplantation at our institution received 10 ABO-unmatched livers. Five (56%) of nine patients developed DDAbs and hemolysis. All five patients were group A1 and received group O livers. DDAbs appeared a mean of 9.2 +/- 2.8 (1 SD) days after surgery and persisted for 15.2 +/- 10.3 days. All patients with DDAbs developed hemolysis. During the period when DDAbs were demonstrable, the hemoglobin dropped by a mean of 4.8 g per dL (48 g/L), and the patients were transfused with a mean of 7.8 +/- 2.3 units of group O red cells. One patient with hemolysis underwent exchange transfusion for acute renal failure. Patients with hemolysis required significantly more red cells postoperatively (15.0 vs. 6.9 units, p = 0.04) than did ABO-matched patients. None of the parameters examined (age, recipient or donor gender, secretor status, rejection, or donor isoagglutinin titers) were predictive of DDAb or hemolysis, although hemolysis occurred in three of four cases in which donor serum IgG anti-A titers were > or = 128, as opposed to one of four cases in which titers were < 128. Because recipients of ABO-unmatched livers are at high risk for transiently developing DDAb and hemolysis with associated morbidity, the prophylactic use of donor-type red cells for surgery and after operation is justified.

IgM autoagglutinins in warm autoimmune hemolytic anemia: a poor prognostic feature.

The presence of both complete IgM autoagglutinins and IgG autoantibodies in warm autoimmune hemolytic anemia (AIHA) is an uncommon finding. Over a 6-year period, only 5 of 115 (4.1%) patients with AIHA had IgM and IgG autoantibodies. In 3 of the 5 cases, the complete IgM autoagglutinins reacted up to 30 degrees C and these patients responded well to corticosteroid or other therapies for warm AIHA. The 2 patients who had warm (37 degrees C) reactive IgM autoagglutinins, were refractory to corticosteroids, splenectomy and cytotoxic drugs, and died due to the complications of hemolytic anemia. The data in these 5 cases suggest that the thermal amplitude of the IgM antibody in these unusual AIHA cases may be predictive of refractoriness to therapy and poor clinical outcome.