What is the mechanism for acute hemolysis occurring in some patients after intravenous anti‐D therapy for immune thrombocytopenic purpura?

In 1983, in the hypothesis section of Lancet, Salama and colleagues1 suggested that the mechanism involved in the successful treatment of immune thrombocytopenic purpura (ITP) with intravenous immunoglobulin (IVIG) was due to contamination of IVIG with red blood cell (RBC) alloantibodies. Their suggestion was that such antibodies sensitize the recipient's RBCs and that the immunoglobulin (Ig)G-sensitized RBCs compete successfully with the IgG-sensitized platelets (PLTs) for Fc receptor sites on macrophages. In this short report they reported on six D+ ITP patients who were given intravenous (IV) anti-D to produce a mild hemolytic syndrome with a positive direct antiglobulin test (DAT); the PLT counts in three of these patients increased considerably. In 1986,2 the same group reported a similar approach to treat 17 ITP patients; significant elevation of the PLT count occurred in 15 of the patients. Other reports from the United States3 and Canada4,5 followed. The Rh Institute in Winnipeg, Manitoba, Canada, had produced an intravenous anti-D (WinRho) for prophylaxis of Rh disease. This product was used to treat ITP in the United States3 and Canada4,5 with good results and few adverse reactions; there was no acute hemolysis reported. The Food and Drug Administration (FDA) licensed Rho(D) immune globulin (anti-D IGIV, then WinRhoSD, currently WinRhoSDF[Cangene Corp., Winnipeg, Manitoba, Canada]) in 1995, for the treatment of ITP in D+, nonsplenectomized children with acute ITP and children and adults with ITP secondary to HIV infection. In 1997, Scaradavou6 reported on the successful results of treating 261 D+ ITP patients with WinRhoSD. The mean hemoglobin (Hb) decrease was 0.8 g/dL (SD1.5) 7 days after the first treatment. No patient required a RBC transfusion. Twenty-nine (16%) had Hb decreases of greater than 2.1 g/dL (10 of these had received multiple infusions with a total dose of greater than 60 µg/kg in the first week). No acute hemolysis was noted. As clinical use widened, a few reports appeared of a more severe hemolytic anemia than expected in some patients. For instance, Barbolla and coworkers7 described a patient who received IV anti-D (15 µg/kg body weight) for 3 consecutive days. After the third dose, on the 14th day, the Hb level decreased from 15.4 to 7.2 g/dL, with a reticulocyte count of 12%, and many erythroblasts were noted on the blood film. The bilirubin level was only 1.9 µg/dL and no hemoglobinemia/hemoglobinuria was mentioned. The authors calculated that the dose of IV anti-D (3000 µg) would have been enough to destroy a maximum of 300 mL of D+ RBCs, but it was estimated the patient had destroyed 1600 mL of RBCs. Seven cases of acute onset hemoglobinuria were reported to the manufacturer of WinRho.8 In 2000 Gaines9 (from the FDA) reported that since 1999 the FDA had reviewed 15 reports of hemoglobinemia and/or hemoglobinuria after administration of WinRho, the only FDA-licensed IV anti-D available at that time. Seven of the patients required transfusion; eight patients developed renal problems (two required dialysis). One patient died as a result of the hemolytic anemia. There have been other reports10-12 of renal insufficiency after treatment of ITP patients with IV anti-D since the report by Gaines.9 A very interesting case report was published in 2004 by Rewald and Francischetti13 where they described an ITP patient treated with IV anti-D minidoses for 8 years (4-5.5 µg/kg body weight with intervals of 10-20 days). For 8 years the treatment was uneventful (Hb remained approx. 8 g/dL). The patient then had marked hemoglobinuria each time after receiving anti-D. This continued for 6 months. Changing the lots/brand of anti-D did not prevent the continuing reactions. No renal problems developed. Treatment with anti-D was stopped, and except for occasional transfusions to treat the continuing pancytopenia, the patient kept stable for the 18 months before the report was published. Mechanisms for the acute hemolysis could not be explained. Three widely different frequency estimates for acute hemolysis with hemoglobinemia and hemoglobinuria were calculated: clinical studies of 528 ITP-treated patients contained no reports; the original clinical trial found 2 in 137 patients (1 in 69); and FDA data (1995-1998) yielded 13 cases in 14,500 treated patients (0.1%, or 1 in 1145 incidence). In 2005, Gaines14 added to the problems encountered by reporting on patients who received IV anti-D for treatment of ITP and developed disseminated intravascular coagulation (DIC) in addition to acute hemolytic anemia. The FDA received six reports of DIC, through November 30, 2004, five of which involved fatalities. I was glad to see that Gaines deliberately avoided the term “intravascular hemolysis,” instead using “acute hemoglobinemia/hemoglobinuria” in her 2000 and 2005 publications.9,14 She discussed her rationale fully in her 2005 article. The distinction between an extravascular and intravascular mechanism of hemolysis was made as early as 1901.15 Intravascular hemolysis associated with an immune hemolytic anemia (e.g., hemolytic transfusion reactions [HTRs] or autoimmune hemolytic anemia) is where RBCs break down directly within the bloodstream, releasing their Hb into the plasma, leading to a reduction in haptoglobins and the presence of free Hb (hemoglobinemia); this Hb will be converted to the brown pigment methemalbumin. When the haptoglobin system is saturated, the Hb may be excreted in the urine (hemoglobinuria). When this type of hemolytic anemia is induced by antibody, damage to the RBCs is caused by activation of the complement cascade, leading to insertion of C5,6,7,8 through the bilipid layer of the RBC membrane, with subsequent entry of water and ions into the RBC, leading to cell rupture. Complement-associated intravascular lysis is rare. Few blood group antibodies activate complement efficiently enough to complete the complement cascade. The most efficient complement activating antibodies are anti-A and -B. All ABO antibodies are capable of activating complement in vitro, but not all cause intravascular lysis in vivo. Other antibodies that can activate complement are anti-Lea, -Leb, -Jka, -Jkb, -Fya, -Fyb, -K, -P, -PP1Pk, -Vel, and -Ena; powerful anti-H; and anti-I/i. Not all examples are capable of activating complement, and most of them are not efficient enough to cause intravascular hemolysis in vivo. Extravascular hemolysis is caused by interactions of cells (e.g., macrophages) within the reticuloendothelial system (RES), in particular, the spleen and liver, and proteins (e.g., IgG1, IgG2, IgG3, IgA, C3b) on the RBC membrane. If these interactions occur in optimal conditions, then several events can occur: phagocytosis of RBCs, partial phagocytosis of RBCs with release of spherocytes, and cytotoxic attack of the RBCs by macrophages; one or more of these events may occur or dominate. Most blood group antibodies (e.g., Rh) destroy RBCs through this mechanism. Haptoglobins are found to be decreased in most, if not all, immune hemolytic anemias, probably because Hb can escape from the RES during the above interactions.16 Thus, Dacie16 argued, many years ago, that “the concept of an absolute distinction between intravascular and extravascular hemolysis, which implies an absolute difference in hemolytic mechanisms, has therefore to be abandoned.” Nevertheless, the two terms are useful and remain in common use. This is acceptable as long as the users realize that hemoglobinemia and hemoglobinuria do not equate with complement-induced intravascular lysis. These signs only suggest that intravascular lysis might be occurring. As mentioned before, Gaines9 obviously concurred with this assessment. Her reports posed several major questions. Is powerful anti-D (e.g., WinRho) sometimes capable of activating complement and causing intravascular hemolysis, renal failure, and DIC? This triad certainly suggests complement activation, but is the culprit anti-D or something else in the product? Why do so few patients receiving this product have such severe hemolysis? In this issue of TRANSFUSION, Gaines and colleagues17 describe a patient with acute hemoglobinemia/hemoglobinuria after administration of anti-D (WinRho). They try to answer the question of whether the anti-D activated complement, leading to intravascular hemolysis, as may occur in an acute HTR. To test for complement activation, they looked for in vitro hemolysis of D+ RBCs in the presence of WinRho and a source of human complement. They tested seven lots of anti-D, four of which had been implicated in acute hemolytic reactions. Their results were negative; even so, they are to be commended for publishing these negative results as authors usually only rush to publish when obtaining positive results. The results, of course, fit with what we know about anti-D but not with the clinical results; even powerful anti-D usually does not activate complement. I know of only three reports of Rh alloantibodies that activated complement, detectable by routine methods. The first18 was an Rh antibody (anti-C+D), famous for its use in the early work on IgG allotypes (it is known as Ripley [Ri]). It was from an immunized male and had a titer of 9 to 18,000 and was found to hemolyze trypsin-treated RBCs. The second19 was an anti-D made by a person with a weak D phenotype. The antibody only had a titer of 16 and reacted strongly with enzyme-treated RBCs and untreated RBCs by indirect antiglobulin test (IAT) using anti-IgG and anticomplement. The third20 was an anti-Rh27 (cE). The antibody appeared to be a naturally occurring IgM antibody; it reacted strongly with cE+ RBCs using anti-C3 by IAT. Rh antibodies should be biologically capable of activating complement as they are usually IgG1, IgG3, or a mixture of both. It was suggested that they did not usually do this as IgG antibodies need to fall close enough together on the RBC membrane to form a “doublet” to activate the first component of complement (C1). IgG anti-A and -B can do this as there are 1 to 2 million A/B sites on A or B RBCs, but there are only 20 to 30,000 D sites on RBCs. Using a C1a fixation and transfer test, Rosse and Parker21 suggested that Rh antibodies could activate C1 if several Rh specificities were mixed (i.e., yielding more sites), but Freedman and colleagues22 could not confirm these findings using radiolabeled antibodies. I also could not confirm that mixtures of Rh antibodies would sensitize RBCs with complement using anti-C3 in an IAT, and I also could not sensitize D+ RBCs with complement using RhoGam (Ortho Clinical Diagnostics, Raritan, NJ) with a titer of 200,000 (unpublished observations). Nevertheless, there are reports that, under certain conditions, anti-D can activate complement. In 1960, Klun and Muschel23 showed that anti-D would fix complement if RBC stroma, rather than intact RBCs, were used. Hidalgo and coworkers24 similarly showed that Rh antibodies would sensitize papain-treated RBC ghosts with complement. They suggested that it was due to aggregation of the Rh sites (yielding “doublets”) under these conditions. Jaffe25 showed that by conventional methods, Rh antibodies showed no complement fixation, but when anti-D–sensitized RBCs were labeled with 51Cr and injected into patients and normal volunteers, the RBCs survived better in C4-deficient patients. In 1981, Hughes-Jones and Ghosh26 showed that C1q would bind to anti-D–coated RBCs provided the density of anti-D on the membrane was sufficiently great. Thus, the inability to activate complement is more likely to be due to failure of activation stages after C1q (e.g., C1r, C1s, C4/C3 activation). The answer is yes. As mentioned previously, plasma haptoglobins are diminished in almost all hemolytic anemias, including HTRs,27 so small amounts of free Hb must be present in the plasma. Haptoglobins can bind 50 to 150 µg of Hb/dL of plasma. One needs approximately 15 µg Hb/100 mL to observe barely visible hemolysis; 45 µg Hb/100 mL will produce pink plasma, and 60-100 µg Hb/100 mL will produce red plasma. Thus, 5 mL of RBCs would have approximately 1650 µg Hb; in a male patient with a plasma volume of 3000 mL, this would represent 55 µg Hb/100 mL, yielding clearly visible pink to red plasma.28 When RBC 51Cr survival studies are performed for studying destruction of Rh-incompatible RBCs, one finds minimal amounts of 51Cr (released from the labeled RBCs) in the plasma, but some (e.g., <5% of the injected amount) is detected.29,30 The article by Jandl and colleagues in 195729 is recommended reading for anyone interested in this subject with relation to ABO and D. Although we do not see many HTRs due to anti-D now, many of us have seen patients with hemoglobinemia/hemoglobinuria after a HTR due to anti-D, usually when more than 1 unit of blood is destroyed. The old literature is educational31,32 as often several units of D+ blood were transfused and hemoglobinuria was quite common. I have never been able to demonstrate complement binding by the anti-D associated with these types of reactions (unpublished observations). So, how does the Hb get into the plasma/urine mimicking complement-mediated intravascular lysis? Experiments in dogs showed that hemoglobinemia can result from “overloading” the RES with damaged sequestered RBCs.33 Now that we understand that at least three events (as discussed earlier) occur in the destruction of sensitized RBCs, it seems possible that Hb could be released when the macrophages partially ingest RBCs and/or a cytotoxic attack occurs. Together with breakdown of the RBCs within the macrophage, the system for dealing with free Hb within the RES could be overloaded and Hb may be released into the plasma. These events would occur completely independent of complement activation, but might produce a similar set of laboratory results. Although the above scenario might explain the hemoglobinemia/hemoglobinuria encountered in HTRs due to non–complement-activating antibodies (e.g., anti-D), I do not think that it would explain the clinical outcomes of the treated ITP patients described by Gaines.9,14 Many of the observed clinical symptoms after the treatment, and the clinical consequences such as renal failure and DIC, suggest the involvement of complement activation. To determine if the IV anti-D associated with hemoglobinuria/hemoglobinemia was activating complement leading to direct lysis of the patient's RBCs, Gaines and colleagues17 used a simple assay looking for direct lysis of D+ RBCs in the presence of complement. There are other simple approaches that could be used such as the IAT using a powerful anti-C3 and testing enzyme-treated RBCs in the hemolysin test. I do not agree with Gaines and coworkers17 when they say that they did not use enzyme-treated RBCs because that would not closely approximate in vivo conditions (“physiologic conditions”). Almost all of the tests we perform in immunohematology do not approximate in vivo conditions. Every day we select blood for transfusion and judge clinical significance by basically adding two drops of serum to one drop of 2% to 5% RBCs, often with potentiators added (e.g., low-ionic-strength saline [LISS], polyethylene glycol [PEG], albumin); these tests are nowhere close to physiologic conditions. When we evaluate drug-induced immune hemolytic anemia, the results correlate well with what we observe in the patients, yet the amount of drug we use in vitro to demonstrate that the patient's antibody causing agglutinates, hemolysis, or sensitization detected by the antiglobulin test in the presence of a particular drug is often nowhere near the amount of drug present in vivo; in fact, penicillin antibodies cannot be detected in vitro if one uses a concentration equal to that in the plasma in vivo, even though the penicillin antibodies are causing the hemolytic anemia and are detected in vitro if much larger quantities of drug are used. “Physiologic” conditions are difficult, if not impossible, to achieve as so many influencing factors are involved in vivo, some of which we do not even know about. I emphasized this in a recent publication34 concerning the gaps in our knowledge of immune RBC destruction. When our routine, or even special approaches fail to explain results in a patient, we must start thinking “out of the box” and try to come up with a mechanism, which is not what we teach or what we learned as a student. Sometimes antibodies can cause intravascular hemolysis without activating complement. Antibodies to determinants on glycophorin A (Pr, Ena) can cause membrane proteins to aggregate allowing Na2+ and Ca2+ to enter the RBCs causing lysis.35 Antigen-antibody reactions can sometimes generate reactive oxygen species (ROS), for example, hydrogen peroxide and ozone, which have been shown to sometimes cause lysis of PLTs,36 so why not RBCs? It is of interest to note that Coopamah and colleagues37 showed that when IgG anti-D–sensitized RBCs were incubated with white blood cells, hydrogen peroxide and other ROS were generated by monocytes and granulocytes. Another report by Lee and coworkers38 showed that mice lacking leucine zipper transcription factor (Nrf), which mediates up regulation of antioxidant detoxification and antioxidant genes in to immune hemolytic anemia. These mice had RBCs of increased IgG by and RBCs that were more to hydrogen lysis. It was suggested that a in from decreased antioxidant RBCs with IgG and hemolytic anemia in a for a in the antioxidant These two the one by Coopamah and suggest a mechanism that could in vivo with blood group hemolysis, but may not be using our routine in vitro HTRs with no detectable antibodies have been reported since the In we reported such patients encountered a of these patients had hemoglobinemia and hemoglobinuria after transfusion of RBCs. of the patients had antibodies detectable by a simple the indirect test three two two one and one These antibodies were not detected by other used tests In eight patients, antibodies could not be but RBCs caused no Sometimes antibodies and detectable by routine methods. Thus, in some we could alloantibodies by certainly and that these antibodies were causing the acute severe HTRs (e.g., blood lacking the caused no In most cases we could not the sometimes blood caused no As one or more units of RBCs were often destroyed within and no complement activation was with no clinical the hemoglobinemia/hemoglobinuria was to be associated with extravascular lysis with the RES not able to with the Hb and the Hb in the plasma and the if in such patients, there is an increased of cytotoxic attack by macrophages. There is no to explain why a small of ITP D+ patients receiving IV anti-D have severe acute hemolytic anemia associated with sometimes renal failure, and on Although these all one of complement activation, so there is in vitro to this some of the discussed in recent publication34 and by and might to the For instance, could the findings be to the presence of antibodies other than IV anti-D, is made by plasma from many and can antibodies to blood group other than D (e.g., other these multiple specificities to complement activation as suggested by Rosse and about the of antibodies to (e.g., antibodies to determinants on plasma of these could activate complement when with the encountered in the and cause lysis of RBCs. with ITP many transfusions (e.g., plasma before they the IV anti-D. There is good that antibodies have caused HTRs associated with intravascular hemolysis for more So, is anti-D the or is some other in the product causing these a few patients to this of no and can we a to for such These are very similar to we have been for drug-induced immune hemolytic anemia have FDA-licensed because clinical do not a significant of patients who after drug yet we a small of who severe sometimes associated with renal failure, and fatalities. A good are the which we have found to be the most common cause of during the few The two are and which were first reported to cause and and and Many reports but the are and we can that drug antibodies are we do not know why patients have such severe hemolysis after receiving these The by Gaines and to the rather long of of immune RBC destruction that we do not It a for The no of

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.