Rh blood group-specific antibodies in immune hemolytic anemia induced by nomifensine

Nomifensine (Merital, Alival; Hoechst, Frankfurt, FRG), an antidepressant drug, may cause immune hemolytic anemia (IHA) of the so- called immune complex type that is believed to occur by means of an innocent-bystander mechanism. In this report we describe findings that are not consistent with this mechanism in a patient with nomifensine- induced intravascular IHA associated with renal failure. In vitro studies showed a transitory positive direct antiglobulin test (DAT) due to IgG, IgM, and C3 fixation. The causative antibodies were found to be a drug-independent IgM antibody in the serum and eluate that reacted only with E-positive RBC, although the patient's RBC were E-negative; an IgG antibody in the serum and initial eluates that showed a stronger reaction with e-positive than with e-negative or Rhnull RBC, but only in the presence of ex vivo antigen (ie, urine containing the drug and all its metabolites); and an IgM antibody in the serum and initially also on the patient's RBC that, in the presence of ex vivo antigen as well as in the presence of known metabolites of the drug, agglutinated all RBC equally strongly, but was hemolytically more active against E- positive than E-negative cells. Within a few days of stopping the drug the hemolysis rapidly resolved without administration of prednisone, the DAT became negative with anti-IgG and anti-IgM, and the drug- independent anti-E disappeared, but both metabolite-dependent antibodies remained detectable in the patient's serum. We conclude that the production and specificity of the causative antibodies in this case were controlled by a larger antigenic site, presumably consisting of the drug and/or its metabolites plus RBC antigens, rather than by epitopes of the drug or metabolites alone.     

On the mechanisms of sensitization and attachment of antibodies to RBC in drug-induced immune hemolytic anemia

The mechanisms of sensitization and attachment of drug-dependent antibodies to RBC in drug-induced immune hemolytic anemias are largely speculative. Nomifensine has been incriminated in causing immune hemolysis in a large number of patients. The hemolysis was usually of the so-called immune complex type, less commonly of the autoimmune type, and more surprisingly, few patients had developed both types of hemolysis. To determine whether nomifensine (metabolite)-dependent antibodies (ndab) exhibit specificity for antigenic structures of RBC membranes, 30 ndab were tested against large panels of RBC with common and rare antigens. We found that only 14 out of 30 ndab were invariably reactive with all cells tested. Nine antibodies were, similar to the majority of idiopathic or drug-induced autoantibodies, not or only weakly reactive with Rhnull RBC. Three antibodies did not react with cord RBC and could be inhibited by soluble I antigen. The remaining four antibodies gave inhomogeneous reaction patterns or were even negative with selected RBC; their specificity could not be identified. On a Scatchard plot analysis of one ndab, a maximum of 173,000 drug-dependent antibodies of the IgG class can specifically bind per RBC in the presence of the drug. Although nomifensine and its metabolites do not attach tightly onto RBC, our results clearly indicate that RBC do not act as "innocent bystanders," but rather serve as a surface for a loose attachment of drugs that possibly cause a subtle structural change in the cell antigens and, by this means, allow in vivo sensitization; and a specific binding of the resultant antibodies. This concept would explain why these antibodies can be directed against drug-cell complexes, against cell antigens alone (autoantibodies), or against both in the same patient.     

用抗体诱发急性红细胞溶血法建立红细胞在体同步衰老模型的可行性研究

在Mohandas工作的基础上,用抗体诱发动物急性红细胞溶血的方法,提出并建立了一种新的红细胞在体同步衰老的模型。对动物红细胞压积、网织红细胞计数等指标连续跟踪观测发现,溶血后红细胞的应激增生过程基本在注射抗体后10d内完成,动物体内的红细胞具有基本相同的年龄特征,说明这一模型是可信     

Autoantibodies and drug-or metabolite-dependent antibodies in patients with diclofenac-induced immune haemolysis

Two patients with acute immune haemolytic anaemia caused by diclofenac are described. Both patients had developed IgG drug-independent autoantibodies and drug-dependent antibodies. The drug-dependent antibodies in one patient reacted with red blood cells (RBC) only in the presence of urine from patients receiving diclofenac (ex vivo antigen) but not in the presence of the drug itself or its known metabolites. This antibody appeared to recognize a trace metabolite which has not yet been identified. The drug-dependent antibodies in the second patient reacted with RBC in the presence of urine as ex vivo antigen as well as in the presence of the drug itself and its main metabolites. Incorrect diagnosis of such cases often is due to the occurrence of autoantibodies and/or unusual drug metabolism and excretion.     

Preservation of Human Erythrocytes in the Liquid State: Biological Results with a New Medium

Abstract. Red blood cells (RBC) were collected with citrate-phosphate-dextrose (CPD) in a blood-pack optimal additive system. After concentration to 90% hematocrit they were diluted with saline-adenine-glucose medium (SAG-RBC), and stored for 35 days. In this work the RBC were stored in the presence of leukocytes. The SAG medium allows RBC conservation during 35 days at +4°C. The adenosine triphosphate (ATP) level of RBC is compatible with their survival. During the first 2 weeks, hemolysis of SAG-RBC was not greater than in CPD blood. Nevertheless, hemolysis reached 1.49% on day 35, and there was a marked increase in RBC osmotic fragility. Scanning electron-microscopic studies of 35-day RBC showed that the majority of them became echinocytes. After incubation in fresh frozen plasma, the RBC recovered satisfactory osmotic resistance and normal disc shape. The post-transfusion viability was normal with >70% recovery after 48 h. The in vivo restoration of 2,3-diphosphoglycerate (2,3-DPG) was rapid in the transfused SAG-RBC, 50% of the initial 2,3-DPG level being restored in 1 h. The in vivo studies proved that the functional quality of these RBC was compatible with their use in transfusion. The most important problem concerns the supernatant hemoglobin level of the SAG-RBC to be used for massive transfusion.     

Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: potential use in transfusion therapy

Activation of complement cascade via the antibody-mediated classical pathway can initiate red blood cell (RBC) destruction, causing transfusion reactions and hemolytic anemia. In the present study, we have assessed the ability of a human recombinant soluble form of complement receptor 1 (sCR1) to inhibit complement-mediated RBC destruction in vitro and in vivo. Using an in vitro alloimmune incompatibility model, sCR1 inhibited complement activation and prevented hemolysis. Following transfusion of human group O RBCs into mice lacking detectable pre-existing antibodies against the transfused RBCs, systemic coadministration of 10 mg/kg sCR1, a dose well tolerated in human subjects for prevention of tissue injury, completely inhibited the in vivo clearance of the transfused RBCs and surface C3 deposition in the first hour after transfusion, correlating with the half-life of sCR1 in the circulation. Treatment with sCR1 increased the survival of transfused human group A RBCs in the circulation of mice with pre-existing anti-A for 2 hours after transfusion by 50%, reduced intravascular hemolysis, and lowered the levels of complement deposition (C3 and C4), but not immunoglobulin G (IgG) or IgM, on the transfused cells by 100-fold. We further identified potential functional domains in CR1 that can act to limit complement-mediated RBC destruction in vitro and in vivo. Collectively, our data highlight a potential use of CR1-based inhibitors for prevention of complement-dependent immune hemolysis.     

Cianidanol and its metabolites bind tightly to red cells and are responsible for the production of auto- and/or drug-dependent antibodies against these cells

Cianidanol ((+)-2-(3,4-dihydroxyphenyl)-3, 5, 7-chromantriol) is a flavonoid which has been associated with severe immune haemolysis by as yet unclear mechanisms. We report six patients who developed haemolysis while receiving the drug. The disorder was episodic in all patients and resolved after discontinuing the drug. The causative antibodies could be demonstrated in all six cases, even when the haemolytic episode was more than 1 year prior to this study. One patient had developed drug-independent IgG autoantibodies, another simultaneously developed autoantibodies and drug-dependent antibodies (ddab) of the IgG class, while the remaining four patients had only ddab of the IgM and/or the IgG classes. All ddab were reactive with red blood cells (RBC) in the presence of the drug and/or its metabolites (ex vivo antigens), and, quite unexpectedly, also with RBC coated with the drug (metabolites) in vitro or in vivo. This reactivity did not change either by preincubating the antibodies with the drug or by adding large amounts of the drug to the mixture of drug-coated cells plus antibody. It seems that the stable association of cianidanol with RBC generates antigenic sites against which a heterogeneous immune response is elicited giving rise to long-lasting drug-dependent antibodies as well as autoantibodies.     

A New Red Blood Cell Cold Autoantibody (Anti-Me)

A new red blood cell (RBC) cold autoantibody (anti-Me) is presented which was detected in a patient with autoimmune hemolytic anemia of ‘cold type’ associated with Waldenström's macroglobulinemia. The autoantibody was of the IgM (Kappa) class, had a moderate titer, and reacted with cord as well as with adult RBC. Its hemagglutinating activity against RBC was markedly increased in the presence of preheated human milk, but not in the presence of simple sugars or preheated casein. The data suggest that the antibody is directed against a new (glycolipid) antigen common to human RBC termed Me antigen (‘milk enhanced’).     

Single-unit transfusions of RBC enzymatically converted from group B to group O to A and O normal volunteers.

Full-unit transfusions of RBC enzymatically converted from group B to group O by treatment with alpha-galactosidase (ECO RBC) to group O and A normal healthy individuals exhibit excellent in vivo survival times (24-hour survival 95.1% +/- 2.3%, T50 36.9 +/- 4.6 days). These results confirm our earlier findings describing ECO RBC in vitro viability and normal in vivo survival time after small-volume infusions. No significant increase in pretransfusion anti-B titer or score is observed in either group O or A subjects provided that sufficient enzyme is used to treat the cells: Cells transfused to group O recipients require higher levels of enzyme (185 to 200 U/mL RBC) than those infused to group A (90 U/mL RBC). Two separate single-unit transfusions of ECO RBC to one group O recipient (4.5 months apart) also survived normally (24-hour survival 96% and 92%, T50 40 and 36 days) and did not increase preexisting anti-B levels in this subject. ECO RBC were not agglutinated or lysed by recipient sera before or after transfusion. Similarly, no antibody development to the alpha-galactosidase used in cell treatment (and washed from the product before transfusion) could be detected in any subject. The sustained increase in hemoglobin levels after transfusion of ECO RBC suggests that this product will be useful in treatment of acute and chronic anemia.     

Allogeneic peripheral blood hematopoietic stem cell transplantation: guidelines for red blood cell immuno-hematological assessment and transfusion practice.Société Française de Greffe de Moelle

Allogeneic peripheral blood hematopoietic stem cell transplantation (PBSCT) is presently being evaluated in a French randomized study comparing peripheral blood vs bone marrow. Cases of potentially lethal acute hemolysis have recently been reported after allogeneic PBSCT in the presence of a 'minor' ABO incompatibility. Patients were frequently transfused with recipient-compatible and donor-incompatible RBC and usually did not receive methotrexate in addition to cyclosporin A for graft-versus-host disease (GVHD) prophylaxis. In order to homogenize immuno-hematological (IH) assessment and transfusion practices within our protocol, we made proposals to 25 allo-transplant French centers on the following aspects: pre-inclusion IH assessment, IH exclusion criteria, transfusion rules, post-transplant IH surveillance and treatment of hemolysis. Analysis of responses to our proposals led to the elaboration of guidelines which were approved and implemented by the French Bone Marrow Transplantation Society (SFGM). Pre-inclusion IH testing includes mandatory detection and titration of anti-RBC allo-Ab, as well as titration of anti-A and anti-B Ab. The presence in the donor of an anti-A (group A or AB recipients), anti-B (group B or AB recipients) Ab with a titer >1/32 or the presence of allo-Ab against Rh, Kell, Fya, Fyb, Jka, Jkb, Ss Ag present on recipient RBC is an exclusion criterion for the protocol. ABO and RhD compatibility of RBC blood products with both HSC donor and recipient is mandatory. A similar compatibility is also required for Rh (other than D) and Kell Ag. If not possible, compatibility of RBC blood products with the HSC donor is mandatory. Lastly, guidelines regarding post-transplantation IH follow-up as well as acute hemolysis treatment have been elaborated. The implementation of these guidelines should contribute to enhancing the quality of transfusion practice after PBSCT. Such an approach will be applied to other aspects of transfusion medicine in the setting of HSC transplantation. Bone Marrow Transplantation(2000) 25, 507-512.     

Morphological alterations and increased resistance to hemolysis in t-butyl hydroperoxide incubated RBC from elderly subjects

The response of human red blood cells (RBC) to oxidative stress has been studied with the aim to evaluate any difference in the behavior of cells from young and old subjects. Thus, RBC from 5 young (27 +/- 2 years) and 5 old (80 +/- 5 years) individuals have been treated with the organic peroxide t-butyl hydroperoxide (TBHP). The two groups behaved differently: after 4 hrs of incubation in 0.5 mM TBHP, RBC from young donors showed a higher level of hemolysis; instead, RBC from old individuals showed abnormal morphologies, being absent in unstressed RBC, with constriction and budding, which could be identified as poikilocytosis. The same abnormal forms are found in patients with spectrin mutation, leading us to hypothesize that TBHP causes damage to the cytoskeletal spectrin. This suggests that poikilocytosis might be an early stage of red blood cell hemolysis because their presence is associated to a lower level of hemolysis.     

High-Titer, High-Thermal-Amplitude Cold Autoagglutinin Not Associated with Hemolytic Anemia

An example of an unusual cold autoagglutinin is reported. The antibody was monoclonal IgMKappa able to fix complement, and, in the presence of albumin, had both a high titer (greater than 4,096 at 4 degrees C) and a wide thermal range (4-37 degrees C). The patient was closely followed over a 3-year period with no evidence of hemolysis ever documented, despite a persistently positive direct antiglobulin test and the presence of the cold autoagglutinin. In contrast to previous reports regarding cold-agglutinin disease, this case demonstrates that in vivo hemolysis is not always associated with cold autoagglutinins that in vitro show a high thermal range in the presence of albumin.     

Donath-Landsteiner Autoimmune Hemolytic Anemia in Children

During the last 4 years, we have studied a total of 531 adults and 68 children with clinically and serologically well-defined forms of immune hemolytic anemias. Among these, Donath-Landsteiner (DL) hemolysis was the underlying disease in 22 of the 68 children (32.4%), but was not observed in adults. All children with DL hemolysis suffered from acute infections presumably of viral origin. In none of the cases was the DL hemolysis suspected clinically. Boys were more often affected than girls. The hemolytic episodes were severe, but resolved within few weeks. Serologically, all patients had a strongly positive direct antiglobulin test (DAT) using anti-C3d reagents, but a weak (n = 6) or negative (n = 16) IgG-DAT. DL hemolysins were always weak and transient, detectable with enzyme-treated red blood cells (RBC) in all, with untreated RBC in 12 of 22 sera. To explore the reason why these weak antibodies can cause extensive hemolysis in vivo, we compared the action of DL antibodies and of cold agglutinins (anti-I) on RBC by several reincubations at 4 and at 37 degrees C. The data obtained from this experiment demonstrate a stronger aggravation of hemolysis by DL than by anti-I antibodies, presumably due to low-affinity interaction between the cells and DL antibodies.     

Antiidiotypic IgG crossreactive with Rh alloantibodies in red cell autoimmunity

IgG autoantibodies eluted from RBCs of antiglobulin positive normal blood donors contained at least two antibody populations, an IgG autoantibody (Ab 1), and an IgG population (Ab 2) that agglutinated RBCs coated with some Rh(D) alloantibodies. Eight of 24 autoantibody eluates tested agglutinated 3 of 10 anti-Rh(D) sensitized RBCs. The agglutinating activity was inhibited specifically by preincubation of the autoantibody eluate with the reactive anti-D. The reaction did not require the Fc domain of the anti-Rh(D), since autoantibody eluates agglutinated RBCs coated with F(ab')2 prepared from the reactive anti-D sera. These findings indicate that the RBCs of some antiglobulin- positive blood donors contain an immunoglobulin auto-antiidiotype (Ab 2) against the RBC autoantibody (Ab 1) which is demonstrable through its cross-reactivity with selected Rh(D) alloantibodies. Identification of auto-antiidiotypes in RBC autoimmunity lends support to the idiotype- antiidiotype network hypothesis of immune regulation and is consistent with the bizarre and complex serology of autoimmune hemolytic anemia. The absence of clinical hemolysis in antiglobulin-positive normal blood donors suggests that immunoglobulin idiotype-antiidiotype interactions may play a role in modulating the effects of RBC autoimmunity.     

High prevalence of Dapsone-induced oxidant hemolysis in North American SCT recipients without glucose-6-phosphate-dehydrogenase deficiency

Dapsone (4-4'-diaminodiphenylsulfone) is commonly used for Pneumocystis jirovecii pneumonia (PCP) prophylaxis in immunocompromised patients. Oxidant hemolysis is a known complication of dapsone, but its frequency in adult patients who have undergone a SCT for hematological malignancies is not well established. We studied the presence of oxidant hemolysis, by combining examination of RBC morphology and laboratory data, in 30 patients who underwent a SCT and received dapsone for PCP prophylaxis, and compared this group with 26 patients who underwent a SCT and received trimethoprim-sulfamethoxazole (TMP-SMX) for PCP prophylaxis. All patients had normal glucose-6-phosphate dehydrogenase (G6PDH) enzymatic activity. In SCT patients, dapsone compared with TMP-SMX for PCP prophylaxis was associated with a high incidence of oxidant hemolysis (87 vs 0%, P<0.001), and the morphological evaluation of oxidant hemolysis correlated well with laboratory evidence of hemolysis. Dapsone-induced oxidant hemolysis in SCT patients is 20-fold higher than the reported rate in the population of HIV-infected patients, and thus much higher than the prevalence of G6PDH variants in the general population. In our patients, it manifested clinically as a lower Hb that was not significant enough to result in increased packed RBC transfusions.     

Donor-derived red blood cell antibodies and immune hemolysis after allogeneic bone marrow transplantation

Six cases of immune hemolytic anemia attributed to donor-derived red cell antibodies after allogeneic bone marrow transplantation (BMT) are reported. In 2/6 cases, severe intravascular hemolysis was seen, 6/6 required increased red cell transfusion, and 1/6 was treated by plasma exchange. All recipients were receiving cyclosporine to prevent graft-v- host disease. Investigations showed that in each case, the donor lacked ABO or Rho(D) red cell antigens present in the recipient. The direct antiglobulin test was positive in 6/6. Relevant serum antibody (anti-A, four cases; anti-B, one case; anti-D, one case) was first detected one to three weeks after BMT. Eluates made from recipient red cells showed the same specificity as serum antibody. Maximum hemolysis occurred nine to 16 days after BMT, suggesting that active production of antibody by "passenger" donor lymphocytes was the likely mechanism of hemolysis, rather than passive transfer of antibody in the marrow infusion. Retrospective analysis of 21 consecutive cyclosporine-treated BMT patients receiving marrow lacking ABO or D antigens present in the recipient showed that (1) 15/18 patients tested had red cell antibody production against recipient red cell antigens; (2) despite the frequent presence of antibody specific for recipient red cell antigens, only 3/21 patients developed clinically significant hemolysis; (3) clinical hemolysis could not be predicted by donor or recipient red cell antibody titers. We conclude that although red cell antibody against recipient antigens is frequently produced after minor ABO and D mismatched BMT in cyclosporine-treated recipients, only 10% to 15% of cases develop clinically significant immune hemolysis. The data presented show that the most likely source of antibody is "passenger" donor lymphoid cells.     

Degradation of glycophorin A of human erythrocytes in patients with myelo- or lymphoproliferative disorders: possible role of neutrophil proteases

We have previously reported that glycophorin A (GPA) of human erythrocytes (carrying blood group M and N determinants) was totally digested by incubation of erythrocytes with human neutrophil elastase (HNE) and cathepsin G (CathG). The membrane-bound GPA fragments fractionated by SDS-PAGE gave characteristic patterns of bands detected by immunoblotting with the monoclonal antibody PEP80. Erythrocytes were incubated with HNE and CathG at low enzyme concentrations, similar to those found in vivo . Characteristic electrophoretic patterns of bands derived from a partial GPA digestion were observed and these patterns were different for both enzymes and different from those obtained after total GPA digestion. GPA was also partially digested by incubation of erythrocytes with granulocytes in the presence of Ca²⁺ and calcium ionophore and electrophoretic pattern of digestion products was similar to that obtained with low doses of HNE. No GPA digestion products were detected after treatment of erythrocytes with plasmin and kallikrein. Untreated erythrocytes of 21 patients with various myelo- or lymphoproliferative disorders were tested by SDS-PAGE of RBC membranes and immunoblotting with the anti-GPA PEP80 antibody. GPA degradation products, resembling those formed by a mild CathG treatment of control RBC, were detected in nine patients. GPA fragmentation was in some cases accompanied by a reduced expression of blood group MN determinants. No distinct relation was observed between the occurrence of GPA degradation in erythrocytes and increases in plasma concentrations of HNE-α₁-proteinase inhibitor (α₁-PI) complex considered to be an indication of a release of neutrophil proteinases in vivo . However, the results suggested that a partial GPA degradation in haematological proliferative disorders may occur due to limited proteolysis by neutrophil proteinases, most likely by CathG.     

Antioxidants in sickle cell disease: the in vitro effects of ascorbic acid

The authors examined the ability of antioxidants to prevent in vitro oxidant damage to the sickle red blood cell (RBC). One millimolar ascorbic acid and alpha-mercaptopropionylglycine significantly (p less than 0.005) protected against RBC Heinz body formation during incubation with acetylphenylhydrazine, while cysteine, cysteamine, and methionine did not. The effect of ascorbic acid was concentration dependent with concentrations as low as 0.1 mM having significant antioxidant effects. Ascorbic acid protected the RBC against hydrogen peroxide induced hemolysis as well (p less than 0.05). Ascorbic acid had a significant stimulatory effect on the rate of glucose oxidation by the pentose phosphate shunt (PPS), especially in the sickle RBC. Ascorbic acid did not protect the RBC from a patient with chronic hemolytic anemia due to G6PDTorrance from Heinz body formation, suggesting that an intact PPS is necessary for ascorbic acid to express its antioxidant properties. These data suggest that clinical trials should be undertaken to examine the efficacy of ascorbic acid in the treatment of SCD.     

Non-ABO red blood cell alloantibodies following allogeneic hematopoietic stem cell transplantation

Immune-mediated hemolysis is a well-recognized occurrence which complicates the period following a bone marrow transplant (BMT). However, although many studies have investigated the hemolytic anemia following ABO-incompatible BMT, data regarding the occurrence of alloantibodies against red blood cell (RBC) antigens other than ABO in patients undergoing hematopoietic stem cell transplantation are limited. In this review, we briefly analyze the most important non-ABO red blood cell (RBC) antigen systems involved in the development of post-BMT alloimmune hemolytic anemia, paying particular attention to the pathogenic mechanisms and the clinical significance of the alloantibodies involved. Among the non-ABO RBC antigens, RhD antigen is the one most frequently implicated in the development of post-BMT alloimmune hemolytic anemia. Although less frequent than hemolysis following transplants with ABO incompatibility, non-ABO-incompatible allograft hemolysis may severely complicate the post-BMT period creating difficult clinical management issues. For this reason, we advise careful pre-transplant donor and recipient checks for the most important RBC antigen systems and close post-BMT immunohematological monitoring in those patients undergoing allogeneic hematopoietic stem cell transplant with RBC antigen incompatibility.     

Amphotericin B-induced oxidative damage and killing of Candida albicans

Amphotericin B (AmB) is known to bind to ergosterol in fungal cell membranes, but the precise mechanism of its toxicity to cells is as yet poorly understood. AmB autooxidizes, and it is possible that its antifungal effects could result from oxidative damage. Exposure of protoplasts of Candida albicans to AmB under hypoxic conditions reduced protoplast lysis by as much as 80% compared with incubations in air. Protoplasts were protected from AmB-induced lysis by exogenous catalase and/or superoxide dismutase (SOD). Whole cells of C. albicans were protected by exogenous catalase from AmB-induced leakage of [3H]leucine and from killing by AmB. Cells grown on medium inducing high levels of endogenous catalase were resistant to AmB-induced growth inhibition. In contrast, AmB-induced K+ leakage was not hindered under hypoxic conditions or in the presence of catalase or SOD. Thus the lethal and lytic effects of AmB on C. albicans cells and protoplasts, but not prelethal AmB-induced K+ leakage, are mediated by oxidative damage.