Review: immune hemolytic anemia and/or positive direct antiglobulin tests caused by drugs
G. GARRATTY
In 19851and 19892I reviewed, in this journal, the cur- The Immune Response to Drugs rent viewpoints on drug-induced immune hemolytic ane- To explain the mechanisms involved in positive DATs, mia w)and/or positive direct antiglobulin tests (DATs). and possibly IHA, one has to know what epitopes the At the end of my 1989 review I wrote, "It seems to me drug-induced antibody is directed against. Although that in 1989 we still have more questions than answers. very little new data have emerged since my last review Perhaps we will have more answers when I rereview the in this journal in 1989,2wider reading of the pertinent subject in 1994." Although more drugs causing immune literature convinces me even more that putative anti- aberrations have been added to the list (Table 1),not bodies can be formed only if the drug binds, however much data have emerged since the 1989 review to prove loosely, to the cell (e.g., red blood cell [RBC], granulo- or disprove the theories postulated to explain the in vitro cyte, or platelet) surface. Table 2 lists drugs that have and in vivo findings.Nevertheless, sufficientnew data been shown to bind well to RBCs, that is, the drug bind- have emerged to warrant an update at this time. ing withstands multiple washes in vitro. Thus, it is easy Table 1. Drugs and other products that have caused immune hemolytic to explain formation of drug-RBC membrane protein anemia (IHA) and/or positive direct antiglobulin tests (DATs). conjugates that would provide the basis for antibody for- Acetaminophen Diethylstilbestrol Nafcillin mation through the classic haptenic mechanism. Most Amphotericin B Diglycoaldehyde Nomifensine Ampicillin Dipyrone p-aminosalicylicacid drugs causing positive DATs and IHA do not bind to Antazoline Erythromycin Penicillin G RBCs efficiently, but it is possible, and I believe proba- Aspirin Fenoprofen Phenacetin ble, that they do bind loosely (i.e., noncovalently) to Butizide Fluorescein Podophyllotoxin Carbenicillin Fluorouracil(5-FU) Probenecid RBCs in vivo. Although the classic concept of an Carbimazole Fluorsemide Procainamide immune response to low-molecular-weight substances Carbromal Glafenine Pyramidon Cefamandole Hydralazine Quinidine (e.g., drugs) involves covalent binding, exceptions have cefazolin Hydrochlorothiazide Quinine been described. The Forssman hapten, for example, Cefotaxime 9-Hydroxy-methyl- Ranitidine Cefotetan ellipticinium Rifampicin becomes immunogenic upon binding to a carrier Cefoxitin Ibuprofen Sodium pentothal through noncovalent binding.3 Nucleic acids and Ceftazidime Indene derivatives streptomycin Ceftriaxone (e.g., sulindac) Sulfonamides oligonucleotides, which are acidic polymers, can also Cephalexin Insulin Suprofen become immunogenic, when complexed to basic pro- Cephaloridine Isoniazid Teniposide Cephalothin Latamoxef Tetracycline tein carriers through multiple salt linkages.3 Chlorinated Levodopa Thiopental Thus, if drugs bind to RBCs, however loosely, it is hydrocarbons Mefenamic acid Tolbutamide (insecticides) Melphalan Tolmetin Chlorpropamide Methadone Triamterene Table 2. Drugs that have been shown to combine with the red blood cell Chlorpromazine Methicillin Trimellitic anhydride membrane in vitro efficiently enough lo withstand multiple washes Cianidanol Methotrexate Unasyn† Penicillins (most) Cisplatin Methyldopa Zomepirac Cephalosporins (some) Cyclofenil Methysergide Cisplatin *Drugs were included only when reasonable evidence was presented in the Carbimazole literature to support the conclusion that they caused the immune reaction Carbromal There are many more reported, but the evidence that they caused a positive Cianidanol DAT or drug-induced IHA is often minimal or is totally lacking. Erythromycin Streptomycin †This is B product name for ampicillin sodium + sulbactam sodium. All Tolbutamide other drugs on the list are listed by their chemical, not trade. name.
IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 41 G. GARRATTY
possible that a haptenic response may occur. When the ly, activating complement and leading to thrombocy- hapten (e.g., the drug) combines with a macromolecule topenia. This “immunecomplex” theory was extended (e.g., RBC membrane components), several epitopes to RBCs to explain drug-induced IHA due to drugs other may be created.4,5 Some epitopes would be only on the than the penicillins and methyldopa.15-17 drug (or a metabolite of the drug). Some epitopes may In 1958, penicillin antibodies were discovered. Ley et be so-called “neoantigens”or ‘new antigenic determi- found that a patient’s serum reacted with RBCs nants” (NADs) and would be composed of part drug, from a pilot bottle of a unit of blood, but not with RBCs part membrane components. It is also possible that the from the unit itself. The reaction was due to the then- drug may change the normal membrane components, common practice of adding penicillin to the pilot bottle creating further diversity in the spectrum of neoantigens to reduce bacterial growth resulting from contamination formed. The possibility of this haptenic response for all during frequent sampling for crossmatching purposes. drugs bas been used to explain the mechanisms involved This showed that penicillin-coatedRBCs were easy to in the serologic and clinical findings associated with prepare in vitro and could be used to detect IgM and drug-induced immune cytopenias. It has led to a unifying IgG penicillin antibodies. Soon after, several cases of hypothesis to replace the three suggested mechanisms penicillin-induced IHA were described, and some years (i.e., drug adsorption, immune complex, and autoanti- later cases of penicillin-induced immune thrombocy- body) that have been used for some years.6,7 topenia and granulocytopenia were described.l9 This finding added another possible mechanism for drug- Proposed Mechanisms to Explain Positive induced cytopenia to those mechanisms already DATs and IHA Caused by Drugs described.8-14 This second mechanism, called the Ackroyd8 suggested that a drug (allylisopropylacety- “drug-adsorption”mechanism by Garratty and Petz,I6 lurea [Sedormid/Apronal]), acting as a hapten, con- suggested that RBCs became coated with penicillin in ferred new antigenic properties on platelets, leading to vivo, and that if IgG penicillin antibodies were present antibodies that reacted with drug only when it was they would react with the RBC-bound penicillin, lead- bound to the cell membrane. In 1952, Miescher and ing to IgG sensitized RBCs. This situation would lead to Meischer9 suggested an alternative theory: drug anti- a positive DAT and possible destruction of the IgG-coat- bodies might initially be formed against the drug, and ed RBCs by macrophages. then these antibodies might then react with the drug, In 1966, it was shown that drugs (e.g., methyldopa) forming drug-anti-drug immune complexes. These could cause the production of true RBC autoantibod- complexes could attach nonspecifically to platelets, ies.20 This added a third possible mechanism for drug- leading to their destruction by macrophages. In later induced positive DATs and IHA. A fourth mechanism, publications, Miescher et al. presented some experi- called the “membrane modification” mechanism by mental work in animals to support this Garratty and Petz,16 was suggested when it was shown Shulman12-14 also criticized Ackroyd’s hypothesis. He in 1967 that cephalothin,21,22and later some other proved that drugs such as quinine and quinidine did not drugs (e.g., diglycoaldehyde [INOX] and cisplatin),23-25 bind firmly to platelet membranes because quinidine can affect the RBC membrane so that proteins become could be removed by a single washing of the platelets. bound to the RBCs nonimmunologically. This mecha- He reported that concentrations of drug on the order of nism may lead to a positive DAT, but so far has not been 1 million times the concentration of membrane sites for proven to cause hemolytic anemia. antibody fixation did not interfere with antibody reac- tions. Using equilibrium dialysis, Shulmanalso showed Criticisms of the immune complex theory that the association between cells and drugs was much The so-called immune complex mechanism has come too weak to account for the large amount of antibody under ever-increasing attack in the last few years. Some that the same cells adsorbed; however, drug antibodies of the reasons for the search for an alternative theory are were shown to combine efficiently with drug in the that- absence of cells. Thus, he suggested that patients make 1. No one has clearly demonstrated drug immune an antibody against a stable complex of the drug with complexes on platelets, granulocytes, or RBCs or in some soluble noncellular macromolecule; when the the serum of patients who have drug-dependent drug is received again, drug-anti-drug immune com- antibodies. plexes form and these attach to platelets nonspecifical- 2. Drugs, such as quinine and quinidine, that were
42 IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 Drug-induced red cell problems
thought to react by this mechanism cause throm- binding of quinidine-dependentantibody is consis- bocytopenia in some patients and hemolytic anemia tent with this possibility. in others. It is unusual for both conditions to occur Increasing numbers of drug-dependent anti- together, and it is difficult to explain why the drug bodies reacting with RBCs are being reported to immune complexes would be so selective in indi- have blood group specificity6,28,35-45 (Table 3). In vidual patients. addition, there are several examples of ,antibodies to Quinine and quinidine antibodies bind to platelets chemicals that have shown blood group specificity by their Fab domains,26,27 suggesting specificbind- Beck et al.46 and Dube et al.47 described caprylate- ing rather than the nonspecific binding of immune dependent "albumin" autoagglutinins with anti-c complexes, as originally suggested. This finding also and anti-e specificity.Reviron et al.48described an suggests that the antibodies must be reacting with anti-I autoagglutinin that was enhanced in the pres drug on the platelets or with neoantigen created by ence of sodium azide. In 1982, we described an anti- the drug interaction with membrane components. Jka that at first appeared to be a low-ionic strength Some drug-dependent antibodies show specificity saline (LISS) solution-dependent antibody.*9 It was associated with well-defined platelet and RBC anti- later determined that the reactivity was indepen- gen~.~~.~~Quinidine- and quinine-dependent anti- dent of ionic strength but dependent on a preser- bodies reactive with platelets appear to react with vative (paraben) added to the commercial LISS. specific epitopes on platelet membrane glycopro- Paraben is a methyl ester of hydroxybenzoic acid. teins GPIb/IX and/or GPIIbAIIa. In 1978, Kunicki et The anti-Jka would only react with Jk(a+) RBCs showed that quinine and quinidine-dependent when methyl esters of hydroxybenzoic acid were antibodies failed to react with platelets from added to the patient's serum. The Jk(a+) patient had patients with the Bernard-Soulier syndrome. This no signs of hemolytic anemia and was transfused finding was confirmedby others.31,32 Platelets from with Jk(a+) RBCs with no ill effects.49Judd et al.50 patients with the Bernard-Soulier syndrome are reported three more examples of similar autoanti- known to be deficient in the GPIb/IX complex, bodies. GPV; and at least one other protein. Several investi- gators produced evidence that quinine and quini- Table 3. Drug-dependentantibodies showing blood groupspecificity. dine-dependent antibodies react with epitopes on Nonreactive or weaker- the GPIb/IX and GPIIb/IIIa complex. Visentin et Drug (number of patients) reacting phenotype Reference al.33 studied sera from 13 patients with quinidine- or quinine-induced thrombocytopenia. They found that 10 of 13 sera contained IgG antibodies specific for both GPIb/M and GPIIb/IIIa, two sera reacted with GPIb/IX alone, and one serum reacted with GPIIb/IIIa alone. In all cases, the drug was required Ellipticin, (1) P- 37 for binding of IgG to target glycoproteins. Quinidine Rifampicin(5) Lu(a-b-) 31 and quinine consist of linked quinoline and quinu- Choropmpamide (1) Jk(a-) 38 Sodium thiopental (1) iadult, icord 39 clidine ring structures and are lipophilic molecules. Glafenine(1) Koo 6 Visentin et al.33 suggested that as such molecules Glafenine (1) e- 6 Nomifensine (9 of 16) Rhnull 40 are known to accumulate at amphophilic surfaces, Nomifensine (3 of 16) icord 40 they might concentrate preferentially in hydropho- Fluorouracil(5-FU) (I) icord 41 bic pockets within the chymotryptic-resistant por- Rifampicin (1) icord 42 Tolmetin (1) Rhnull, e- 43 tion of the GPIIIa molecule, where they might Tolmetin (1) Rhnull, e- 44 induce structural changes (neoantigens) that are Sulindac (1) RhnulI, D-- 45 immunogenic in certain individuals. They suggest- ed that antibody bound to such determinants might 5 There have been an increasing number of reports of stabilize the drug-GP complex so that the drug is not patients with sera that exhibit the characteristics of readily dissociated from the molecule by washing. more than one mechanism (e.g., the serum reacts The findingsof Christie et al.34 that tritiated quini- with drug-coated RBCs and also with untreated dine is stabilized in the platelet membrane by the RBCs in the presence of drug, or even reacts with
IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 43 G. GARRATTY
untreated RBCs without the presence of bodies (i.e., those associated with warm AIHA). Mueller- drug).43-45,51-72Many of these reports, concerning Eckhardt and Salama7,73 believed that only one hypoth- not only drugs and metabolites but also insecticides esis is necessary to explain all the phenomena observed. and fluorescein dye, have been published in the last They not only criticized the immune complex hypothe- 5 years, but it is interesting to note that single-case sis but also the drug-adsorption mechanism15-17 and the reports were present in the literature as early as theory that some drugs (e.g., methyldopa and pro- 1959.51 cainamide) cause autoantibody production by directly affecting the immune system.74 A unifying hypothesis incorporating three It is tempting to draw cartoons as Habibi did in refer- mechanisms into one mechanism ence 6, and as I did in references 27 and 75, to illustrate To explain why the sera of four patients with drug- how the immune complex, drug adsorption, and autoan- induced IHA showed the characteristics associated with tibody mechanisms could be explained based on classi- the “immunecomplex” and autoantibody mechanisms, cal hapten immunology. These cartoons are based on the Habibi6 suggested a single mechanism. He suggested findings that in the haptenic response, antibodies can be that following ingestion of a particular drug, the forma- made to the hapten (i.e., the drug), the carrier (e.g., RBC tion of both autoantibodies and drug-dependent anti- membrane components), and/or a population of anti- bodies could be explained by the well-known hapten bodies that may react with part hapten and part mem- and carrier specificities commonly developed in animals brane. One could argue that a patient may make one immunized with hapten-carrier conjugates. He suggest- population or several populations of antibodies showing ed that formation of drug-cell conjugates must be the ini- the different specificities. Thus, an antibody to the drug tial step of most drug-induced cytopenias, and that the alone would react with drug-coated RBCs and be inhih- reason for the rare incidence of this disorder is probably ited by the drug; this would be typical of penicillin anti- that only a few individuals are capable either of coupling bodies, or the “drug adsorption” mechanism. Antibodies drugs to their cells in vivo to form efficient immunogens to membrane components would appear only as an or of mounting an unusually strong immune response to autoantibody. Antibodies directed to epitopes that are such conjugates. Mueller-Eckhardt and Salama7,73 sug- part drug and part membrane would require the drug gested a unifying concept that is basically similar to the and RBCs to be present together before a reaction is concepts of Ackroyds8 and Habibi6 regarding the pro- observed; as most of these drugs do not combine with posed immune response to drugs. They suggested that RBCs efficiently enough to withstand in vitro washing, the immune process is always initiated by a primary one cannot prepare drug-coated RBCs, and the only way interaction of the drug and/or its metabolites with con- of demonstratingsuch antibodies is to mix the patient’s stituents of blood cell membranes. This interaction pro- serum with drug and RBCs. This reaction is typical of the vides the composite antigenic structure, which so-called immune complex mechanism, but if the above provokes the production of two types of antibodies: concept is correct the antibody is reacting with a drug-dependent antibodies and/or drug-independent “neoantigen”formed when the drug binds loosely to cell antibodies. The specificity of drug-dependent antibod- membrane components, rather than to drug immune ies is determined by elements of both the drug and the complexes attaching to the cell. cell membrane (drug-dependent neoantigen). These The unifying concept is attractive because it can be antibodies cannot bind sufficiently well to either one used to explain the Fab-dependent binding of the drug alone. If one part is removed (e.g., by dialysis in vitro, by antibody, the specificity that is sometimes observed, and discontinuance of drug administration, or by subse- the serologic finding suggesting several mechanisms in quent excretion in vivo), the immune reaction subsides. one patient. When the antibody is made against part Drug-independent antibodies are elicited by a subtle drug and part membrane, the proportions recognized alteration of the membrane by the drug, but their bind- as antigenic determinants will vary. For instance, one ing sites are sufficiently similar to, or comprise enough population of antibody molecules may be mainly anti- unaltered structures of, normal blood cell membranes drug, and another population may be mainly antibody to support drug-independent binding to the patient’s directed at membrane (see the cartoons in references 27 cells as well as to normal cells (drug-independent and 75).Thus, it is possible that the membrane compo- neoantigen). Such antibodies behave like autoantibod- nents composing the immunogen will contain specific, ies and cannot be distinguished from “true”autoanti- well-defined platelet or RBC antigens, so the “drug”anti-
44 IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 Drug-induced red cell problems
body will appear to have blood group specificity. that penicillin antibodies may react with preformed Because the proportion of membrane and drug anti- drug-cell complexes, but antibodies to other drugs (e.g., genic determinants will vary, one would expect to see a quinidine and quinine) may react with drugs bound spectrum of serologic activity in different patients. Thus, loosely to the cell membrane, or drug that is not bound patients said to have antibodies showing characteristics to cell membranes. In either case, one could say that a of the immune complex, drug adsorption, and autoan- drug-anti-drug immune complex is formed. To explain tibody mechanisms may have three different antibody the Fab binding of the drug antibody to the cell, populations, but the serologic characteristics may be Shulman and Reid76 suggest that drug-immune com- explained by a single mechanism. plexes could attach to the cell by the weak attraction of Although the above hypothesis was attractive enough the drug to the cell so that the preformed drug immune for me to make a cartoon27.75 to expand on the complex could attach to the cell, or the antibody might Ackroyd/Habibi/MueIler-Eckhardthypothesis, I now bind to drug loosely bound to the cell. If the drug anti- have some reservations about its validity. The hypothe- body also recognizes some membrane antigens (e.g., sis is based on classical hapten immunology studies per- neoantigens), then the binding of the complex to the formed by injecting small-molecular-weight substances, membrane would be stronger. Finally, Shulman and conjugated in vitro to heterologous proteins (e.g., albu- Reid76 suggest that conformational changes in the Fab min) into animals. In a recent review in which they pro- portion of the drug antibody may occur following drug pose a variation on the original immune complex binding, and that such conformational changes may rec- hypothesis, Shulmdn and Reid76 point out that when ognize membrane sites. Such interactions could convert autologous or homologous protein, instead of heterolo- a low-affinityreaction (i.e., antibody reacting with loose- gous protein, is used as a carrier, the complex is less ly bound drug) to a higher-, but still low-, affinity immunogenic and autoantibodies rarely develop. Thus, bimolecular association. If two of the drug antibody‘s Fab the claims that the “unifying”hypothesis is supported sites
IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 45 Personal Opinions of Current Theories drug antibody reacts with the drug-coated RBC. I see My first opinion is that we are still not certain how nothing wrong with continuing this terminology, but drug antibodies are made and why they show specific could readily accept an alternative term to describe the serologic and hematologic (clinical) characteristics. mechanism. I do not like the term “hapten mechanism” Nevertheless, I believe we are coming closer to agreeing because it could be applied to all drugs. that some form of the unifying hypothesis is acceptable Whether we should continue to use the term for explaining the immune response and the serologic “immunecomplex mechanism” to explain the way that characteristics associated with penicillin-type antibodies drug-dependent antibodies other than pencillin react is and nonpenicillin-type drug-dependent antibodies, or open to debate. Some workers feel strongly that, the so-called immune complex group. The hypothesis because of the recent criticisms of the theory, we can also be used to explain the occurrence of apparent should not continue to use the term. I have an interme- autoantibodies appearing together with characteristics diate view and feel that as no other theory has been of other mechanisms (e.g., as seen with the second- and proven to be correct, it is acceptable to use such terms third-generation cephalosporins), but I cannot accept as “the so-called immune complex mechanism,” or to this as an explanation of autoantibodies when they put the words “immunecomplex” in quotes to indicate appear alone, for example, when they are associated that you realize that that mechanism is not a proven fact. with methyldopa or procainamide. I mentioned earlier It is, of course, perfectly acceptable to use the terms that the classical hapten experiments were performed immune complex “hypothesis” or “theory”without with heterologous protein carriers, and we are usually adulteration. It is difficult for me to completely reject dealing with autologous protein carriers. It is interesting the possibility that drug immune complexes sometimes to note that many of the patients with drug antibodies form, as the serologic (e.g., very small amount of drug have been transfused with blood products, and thus may necessary for in vitro reactions; complement activation, have allogeneic proteins circulating at the time they leading to lysis of RBCs in vitro) and clinical (acute receive the drug. It is possible that such allogeneic pro- intravascular hemolysis and sometimes renal failure fol- teins may provide an immunogenic basis somewhere lowing ingestion of very small quantities of drug) find- between heterologous and autologous protein carriers ings are so typical of immune complex-mediated (Patricia Arndt, personal communication, 1994). reactions. However, I like many aspects of the unifying Most investigators would agree that drug antibodies hypothesis. Thus, ut thepresent time, I find Shulman can be classified as drug independent, that is, drug is not and Reid‘s recent suggestions76very palatable, because needed to demonstrate reactivity (Le., autoantibodies), they appear to embrace the best of both approaches. or as drug dependent, where presence of drug is need- ed to demonstrate reactivity. I prefer to divide the latter Problems with Second- and into “penicillin-type”and “nonpenicillin-type”antibod- Third-Generation Cephalosporins ies. I do this because the serologic and clinical charac- In my last review for this journal,2I mentioned a new teristics of these groups are so different (Table 4). phenomenon that serologists started observing in the The penicillin-type drugdependent antibody was said late 1980s. The first-generation cephalosporins (e.g., to react by a “drug adsorption” mechanism, implying cephalothin) rarely caused IHA and, when they did, IHA that drug is adsorbed onto the RBC membrane and the appeared to be through a penicillin-type mechanism. In Table 4. Charcteristics of drug-dependentantibodies the late 1980s, several cases of IHA associated with sec- Penicillin Type NonpenicillinType ond- and third-generation cephalosporins were described.65 These patients had acute intravascular com- 1. Antibody reacts with Drug-coatedRBCs cannot drug-coatedred blood cells (RBCs). usually be prepared in vitro, 80 plement-mediated hemolysis. Since that time, many antibody can only be demon- other similar cases have been described, and I know of strated by testing serum in presence of drug and RBCs. at least six fatalities due to drug-inducedhemolysis, only 2. Antibody can be inhibited Antibodycannot be inhibited three of which have been published.19 Most of the cases by drug (see ten). by drug. have serology that has characteristics of penicillin-type 3. RBC destruction is usually RBC destruction is often together with nonpenicillin-type (‘‘immunecomplex”), not associated with associated with acute intravascular hemolysis intravascular hemolysis, and drugdependent reactions; some sera also appear to con- hemoglobinemia,hemoglobinuria) sometimes renal failure. tain drug-independent antibodies. These cases have been or renal failure. reviewed in more detail elsewhere.19 It is inter-
46 IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 Drug-induced red cell problems
esting to note that several of these cases were sent to of a 1 percent albumin solution helped them get drugs the blood bank to be investigated as possible hemolytic into solution. If there is a convincing history incrimi- transfusion reactions because the hemolytic anemia nating a particular drug and the DAT is positive but the appeared following surgery; cephalosporins are used drug workup is nonproductive, one must remember to prophylactically for certain surgical procedures. IHA has test the serum (and eluate) against metabolites of the now been associated with most of the second- and third- drug (e.g., urine from patients taking the drug).81 generation cephalosporins that are used intravenously The cephalosporins are associated with problems not (cefamandole, cefotaxime, ceftriaxone, ceftazidime, encountered with many of the other drugs listed in Table cefoxitin, and cefotetan). Cefotetanis the chief offend- 1. Most immunohematologists are aware that cephalo- er at present, with six cases in the literature and several thin (Keflin, Eli Lilly, Minneapolis, MN) can modify the unreported cases that we have investigated. RBC membrane so that proteins are adsorbed nonim- munologically. This situation can lead to falsely positive Practical Considerations DATs and also falsely positive indirect antiglobulin tests Regardless of the controversies about the theories on (IATs) when testing sera for cephalothin antibodies. the mechanisms involved in drug immunology, the sero- Thus, a positive IAT using cephalothin-treated RBCs logic approaches remain very much the same. The first means very little if undiluted serum is used. We have pre- point to be emphasized is that drug-induced IHA is rare. viously shown that this nonspecific reaction never A positive DAT is much more likely to be due to causes occurs when the serum is diluted 1:20 or more.82If a other than drug-induced antibodies, even when a reaction occurs when the serum is diluted 1:20 or patient is on any of the drugs listed in Table 1. Even greater, cephalothin antibodies (or cross-reacting peni- when an eluate prepared from the DAT-positive RBCs is cillin antibodies) are probably present. Two questions nonreactive with reagent RBCs, the reason for the non- that have not been answered adequately in the literature reactivity is much more likely to be that the eluate con- are (1) How often does nonspecific adsorption of pro- tains anti-A or -B from out-of-groupplatelet or plasma teins occur with the second- and third-generation transfusions than a drug antibody. One should also cephalosporins? and (2) Are falsely positive reactions remember that up to 80 percent of all positive DATs confined to the antiglobulin test? I recently presented yield nonreactive eluates, and this is thought to be due some data relevant to these questions,” and I believe to nonimmunologic uptake of IgG onto patients’ RBCs that knowledge of these data are important before inter- when their plasma IgG levels are If the eluate is preting serologic results associated with the newer reactive and the serum contains autoantibodies, it is cephalosporins. much more likely that these are associated with idio- Positive IATs due to nonspecific uptake of plasma pathic autoimmune IHA than drug-induced AIHA. proteins onto cephalothin-treated RBCs have been The basic serologic approaches to investigating drug- described in association with cephalothin, cephalexin, induced IHA are the same as described earlier,17,29 but cefazolin, cephapirin, and cefotetan.19 It appears that several technical hints are worth adding or emphasizing. the uptake of proteins can be diminished if the drug- If the characteristics of the putative drug-induced anti- coated RBCs are prepared at a lower pH. In our original body have not been well described in the literature, it is report on optimal methods for preparing penicillin- and wise to test the patient’s serum (and eluate) against cephalothin-treated RBCs, we showed that, in contrast drug-coated RBCs, and against RBCs that have not been to penicillin, cephalothin bound to RBCs as efficiently treated with drug, with and without drug added to the at pH 7.3 and pH 8.2 as at pH 10. Nevertheless, many serum. However, performing drug studies involving workers, including ourselves, found it convenient when new drugs is difficult. If the drug is not listed on Table preparing penicillin- and cephalothin-coated RBCs to 2, it may be difficult to prepare drug-coated RBCs; with- prepare both at pH 8.5-10.0. Branch et al.83 studied sera out a positive control of a known antibody one does not from 113 random patients and found that sera from 97 know if the RBCs are coated, so a negative reaction percent of these patients reacted by IAT when tested means very little. Another problem often encountered against RBCs that had been coated with cephalothin at is getting the drug into solution. It is advantageous to pH 8.5. Only 4.6 percent of the sera reacted against consult the Merck Index79 or the manufacturers of the RBCs prepared with cephalothin at pH 6.0. Thus, it drug, but it must be remembered that the final solution would seem wise to prepare cephalothin-coated RBCs has to be isotonic. Curtis et al.80 reported that the use (and probably other cephalosporin-coated RBCs) at a
IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 47 lower pH than when preparing penicillin-coatedRBCs nation, resulting from an interaction of the patient's (i.e., pH 8.5-10.0); a lowerpH should help considerably serum with cephalothin- or cefotetan-treated RBCs can- in alleviating nonspecific uptake of proteins. One word not be interpreted as indicating the presence of of caution is that Branch et did not provide data to cephalosporin antibodies without performing confirma- show that RBCs were as strongly coated with cephalo- tory studies. This discussion emphasizes that when test- thin at pH 6.0 as at pH 8.5;this information could be ing cephalosporin-coated RBCs, they should always be important in order to maintain sensitivity for detecting tested against control sera from individuals not receiving cephalothin antibodies. Therefore, it might be wise to cephalosporins; such results will help put the positive compromise at pH 7.0-7.3. reactions obtained with serum from a patient with a pos- Another phenomenon not often discussed is that sible cephalosporin-induced cytopenia into perspective. sometimes normal sera agglutinate cephalosporin-coat- ed RBCs; whether this phenomenon is due to the same References mechanism as is postulated for IATs is unclear. Although 1. Garratty G. Drug-inducedimmune hemolytic anemia and/or pot itive direct antiglobulin tests Immunohematology 1985;2:l-8. I can accept that nonspecific uptake of plasma proteins 2. Garratty G. Current viewpoints on mechanisms causing drug- onto cephalosporin-coated RBCs may lead to reactions induced immune hemolytic anemia and/or positive direct with antiglobulin sera, I cannot see why the RBCs would antiglobulin tests. Immunohematology 1989;5:97-106. 3. De Weck AL. Low molecular weight antigens. In: Sela M, ed. Toe be directly agglutinated by sera from individuals who antigens. Val. 2. New York: Academic Press, 1974:141-248. have never received cephalosporins, and who have no 4. Landsteiner K. The specificity of serological reactions. penicillin antibodies. Nevertheless, this phenomenon is Cambridge, MA: HarvardUniversity Press, 1947. commonly encountered with some cephalosporins. In 5. Kitagawa M, Yagi Y, Pressman D. The heterogeneity of com- bining sites of antibodies as determined by specific immunoad- their original report on cephalothin-induced positive sorbents. II. Comparison of elution patterns obtained with DATs, Gralnick et al.22 noted that 17 of 20 (85%) serum anti-P-azobenzoateantibodies by different kinds of immnnoad- samples from healthy individuals caused direct aggluti- sorbent and eluting hapten. J Immunol1965;95:455-65. 6. HabibiB. Drug induced red blood cell autoantibodiesco-devel- nation of cephalothin-coatedRBCs, but only one of these oped with drug specific antibodies causing haemolytic sera agglutinated penicillin-coated RBCs. We recently anaemias. Br J Haematol 1985;61:139-43. tested 30 normal sera with RBCs coated with cephalo- 7. Mueller-Eckhardt C, Salama A. Drug-induced immune cytope- nia: a unifying pathogenic concept with special emphasis on thin and cefotetan at pH 9.5,and found that 97 percent the role of drug metabolites. Trans Med Rev 1990;469-77. and 90 percent, respectively, agglutinated the coated 8. AckroydJF. The immunological basis of purpura due to drug RBCs; 100 percent of the sera reacted by IAT. Far fewer hypersensitivity. Proc R Soc Med 1962;55:30-6. reactions were seen in another experiment, when 30 9. Miescher PA, Meischer A. Die sedormid-anaphylaxie.Schweiz Med Wochenschr 1952;82:1279-82. other normal sera were tested against RBCs coated with 10. Miescher PA, Gorstein F. Mechanisms of immunogenic platelet cephalothin and cefotetan at pH 7.3;RBCs coated at this damage. In: Johnson SA, Monto RW, RebuckJW, Horn RC, eds. pH were agglutinated by 70 percent and 7 percent, Blood platelets. London: Churchill, 1961:671. 11. Miescher PA, Pepper JJ. Drug-induced immunologic blood respectively, of the sera. All of the normal sera reacted dyscrasia: In: Miescher PA, Muller-Eberhard HJ, eds. Textbook by IAT, with RBCs coated with cephalothin at pH 7.3, of immunopathology. 2nd ed. Philadelphia: WB Saunders. but none of the sera reacted by IAT, with RBCs coated 1976421-32. with cefotetan at pH 7.3.We found very few nonspecif- 12. Shulman NR. Immunoreactions involving platelets 1. A steric ic reactions (agglutination or IAT) when testing RBCs coated with cefotaxime or ceftazidime, even when the RBCs were prepared at pH 9.5.Thus, combining our data with those of Branch et it seems that cephalothin and cefotetan lead to a high percentage of falsely positive reactions; cephapirin a moderately high percentage of falsely positive reactions; and cephalori- dine, cefamandole, cephalexin, cefazolin, cefotaxime, and ceftazidimea low percentage of falsely positive reac- tions. It is unclear why cephalosporin-treatedRBCs (e.g., cephalothin and cefotetan) should be directly aggluti- nated by normal sera, even if they have adsorbed normal plasma proteins from the sera. A positive IAT, or aggluti-
48 IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994 Drug-induced red cell problems
20. Carstairs KC, Breckenridge A, Dollery CT, Worlledge SM. Incidence of a positive direct Coombs test in patients on a- methyldopa. Lancet 1966;2:133-5. 21. Molthan L, Reidenberg MM, Eichman MF. Positive direct Coombs tests due to cephalothin. N EngJ Med 1967;277:123-5. 22. Gralnick HR, Wright LDJr, McGinniss MH. Coombs' positive reactions associated with sodium cephalothin therapy. JAMA 1967;199:135-6. 23. Jamin D, DemersJ, Shulman I, et al. An explanation for nonim- munologic adsorption of proteins onto red blood cells. Blood 1986;67:993-6. 24. Jamin D, Shulman I, Lam HT, et al. Production of a positive direct antiglobulin test due to Suramin. Arch Pathol Lab Med 1988;112:898-900. 25. Zeger G, Smith L, McQuiston D, GoldfingerD. Cisplatin-induced nonimmunologic adsorption of immunoglobulin by red cells. ment ofthe Rh factor. Vox Sang 1993;64:179-83. Transfusion 1988;28:1403.5. 46. Beck ML. A fatty-acid dependent antibody with Rh specificity 26. Christie DJ, Mullen PC, Aster RH.Fab-mediated binding of drug- (abstract). In: Book of abstracts from the ISBT/AABB Joint dependent antibodies to platelets in quinidine- and quinine- Congress. Arlington, VA: American Association of Blood Banks, induced thrombocytopenia. J Clin Invest 1985;75:310-4. 1972:6. 27. Smith ME, Reid DM, Jones CE, et al. Binding of quinine- and 47. Dube VE, Zoes C, Adesman P. Caprylate-dependent auto-anti-e. quinidine-dependent drug antibodies to platelets is mediated by Vox Sang 1977;33:359-63. the Fab domain of the immunoglobulin G and is not Fc depen- 48. Reviron M, Janvier D, Reviron J, Lagabrielle JF. An anti-I cold dent. J Clin Invest 1987;79:912-7. autoagglutinin enhanced in the presence of sodium azide. Vox 28. Garratty G. Target antigens for red cell-bound autoantibodies. sang 1984;46:211-6. In: Nance SJ, ed. Clinical and serological aspects of immunohe- 49. Halima D. Garratty G. Bueno K.An apparent anti-Jka reacting matology. Arlington, VA: American Association of Blood Banks, only in the presence of methyl esters of hydroxybenzoic acid 1991:33-72. Transfusion 1982;22:521-4. 50. Judd WJ, Steiner FA, Cochran RK. Paraben-associated autoanti- 29. Christie DJ. Specificity of drug-induced immune cytopenias. Trans Med Rev 1993;VIl:230-41. Jka antibodies. Transfusion 1982;22:31-5. 51. Muirhead EE, Groves M, Guy R, et al. Acquired hemolytic ane- 30. Kunicki TJ, Johnson MM, Aster Absence of the platelet- RH. mia, exposure to insecticides and positive Coombs test depen- receptor for drug-dependent antibodies in the Bernard-Soulier dent on insecticide preparations. Vox 1959;4:277-92. syndrome. J Clin Invest 1978;62:716-9. Sang 52. Hart MN, Mesara BW. Phenacetin antibody cross-reactive with 3 1. Chong BH, Berndt MC, KouttsJ, Castaldi PA. Quinidine-induced autoimmune erythrocyte antibody. Am J Clin Pathol 1969; thrombocytopenia and leukopenia: demonstration and charac- 52:695-701 terization of distinct antiplatelet and anti-leukocyte antibodies. 53. Shulman Arndt PA, McGehee W, Garratty G. Cefotaxime- Blood 1983;62:1218-23. IA, induced immune hemolytic anemia due to antibodies reacting 32. van Leeuwen EP, EngelfrietCP, van dem Borne AEG Kc Studies in vitro by more than one mechanism. Transfusion on quinine- and quinidine-dependent antibodies against platelets 1990;30:263-6. and their reaction with platelets in the Bernard-Soulier syn- drome. BrJ Haematol 1982;51:551-60. 54. Garratty G, Houston M, Petz LD, Webb M. Acute immune 33. Visentin GP, Newman PJ. Aster RH.Characteristics of quinine- intravascular hemolysis due to hydrochlorothiazide. Am J Clin and quinidine-induced antibodies specific for platelet glycopro- Pathol 1981;76:73-8. teins IIb and IIIa. Blood 1991;77:2668-76. 55. Garatty G, Nance S, Lloyd M, Domen R. Fatal immune hemolyt- ic anemia due to cefotetan. Transfusion 1992;32:269-71 34. Christie DJ, Aster RH.Drug-antibody-platelet interaction in qui- nine- and quinidine-induced thrombocytopenia. ] Clin Invest 56. Florendo NT, MacFarland D, Painter M, Muirhead EE. 1982;70:989-98. Streptomycin-specific antibody coincident with a developing 35. Martinez J, Letona J, Barbolla L, et al. Immune haemolytic warm autoantibody. Transhision 1980;20:662-8. anaemia and renal failure induced by streptomycin. Br J 57. Habibi B, Lopez M, Serdaru M, et al. Immune hemolytic anemia Haematol 1977;35:561-71. and renal failure due to teniposide. N Eng J Med 36. Duran-SuarezJR, Martin-Vega C,Argelagnes E, et al. Red cell I 1982;306:1091-3. antigen as immune complex receptor in drug-induced hemolyt- 58. Bird GWG, WinghamJ, Babb RG, et al. Azapropazone-associat- icanemias. Vox Sang 1981;41:313-5. ed antibodies. Vox Sang 1984;46:336-7. 37. Habibi B, Bretagne Y. Blood group antigens may be the recep- 59. Salama A. Mueller-Eckhardt C. Two types of nomifensine- tors for specific drug-antibody complexes reacting with red induced immune haemolytic anaemias: drug-dependent sensiti- blood cells. C R Acad Sci (D) (Paris) 1983;296:693-6. zation and/or autoimmunization. Br J Haematol 1986; 38. Sosler SD, Behzad O, Garratty G, et al. Acute hemolytic anemia 64:613-20. associated with a chlorpropamide-induced apparent auto anti- 60. Salama A, Mueller-Eckhardt C.Cianidanol and its metabolites Jka. Transfusion 1984;24:206-9. bind tightly to red cells and are responsible for the production 39. Habibi B. Hasty R, Chodez S, Prunat A. Thiopental-related of auto- and/or drug-dependent antibodies against these cells. Br immune hemolytic anemia and renal failure. N Eng J Med J Haematol 1987;66:263-6. 1985;312:353-5. 61. SalamaA, Göttsche B, Mueller-Eckhardt C. Autoantibodies with
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50 IMMUNOHEMATOLOGY, VOLUME 10, NUMBER 2, 1994