Drug-induced immune hemolytic anemia associated with anti-vancomycin complicated by a paraben antibody.

BACKGROUND: Drug-induced immune hemolytic anemia (DIIHA) is rare, but potentially life-threatening. A high index of clinical suspicion is required for diagnosis, since the number of medications known to induce DIIHA continues to expand. Additionally, in vitro antibody reactivity against reagent additives has been reported, which may complicate test interpretation. CASE REPORT: A 61-year-old group A, D+ woman with a history of negative antibody detection tests developed hemolytic anemia on Postoperative Day 7 after repeat incision and drainage of a chronically infected right knee prosthesis. She was treated with multiple antibiotics in the postoperative period, including three cephalosporins and vancomycin intravenously as well as vancomycin and gentamicin-containing intraarticular cement spacers. STUDY DESIGN AND METHODS: A workup for possible DIIHA was performed. Testing was performed using vancomycin and cephalosporin antibiotics. Initially, gentamicin injection solution was used for testing, followed by testing with its component ingredients. RESULTS: A vancomycin antibody was detected and anemia resolved after vancomycin was discontinued. Reactivity was seen when gentamicin injection solution was used for testing, raising the possibility of a gentamicin antibody as well. However, testing with purified gentamicin as well as methylparaben and propylparaben demonstrated a paraben antibody that reacted with the paraben-containing gentamicin solution. The patient also demonstrated an anti-N. Neither the paraben antibody nor the anti-N appeared to cause in vivo hemolysis. CONCLUSION: This is the second reported case of DIIHA associated with anti-vancomycin. It is the fourth report describing a paraben antibody.

Utility of chloroquine diphosphate in the blood bank laboratory.

CONCLUSIONS: Chloroquine diphosphate (CDP) is a helpful tool in the blood bank for two main applications. The most common application is to render direct antiglobulin test-positive red blood cells (RBCs) free from membrane-bound IgG; these treated RBCs can then be used for autologous adsorption and/or to determine the patient's RBC phenotype. Another common use of CDP is to remove human leukocyte antigens (HLAs) from RBCs to help identify or exclude the presence of antibodies to HLAs expressed on RBCs, for example, Bennett-Goodspeed (Bg) antigens. In this review, the principles, applications, and limitations of using CDP are discussed.

First example of an FY*01 allele associated with weakened expression of Fya on red blood cells.

Duffy antigens are important in immunohematology. the reference allele for the Duffy gene (FY) is FY*02, which encodes Fy(b). An A>G single nucleotide polymorphism (SNP) at coding nucleotide (c.) 125 in exon 2 defines the FY*01 allele, which encodes the antithetical Fy(a). A C>T SNP at c.265 in the FY*02 allele is associated with weakening of Fy(b) expression on red blood cells (R BCs) (called Fy(x)). until recently, this latter change had not been described on a FY*01 background allele. Phenotype-matched units were desired for a multi-transfused Vietnamese fetus with α-thalassemia. Genotyping of the fetus using a microarray assay that interrogates three SNPs (c.1-67, c.125, and c.265) in FY yielded indeterminate results for the predicted Duffy phenotype. Genomic sequencing of FY exon 2 showed that the fetal sample had one wild-type FY*01 allele and one new FY*01 allele with the c.265C>T SNP, which until recently had only been found on the FY*02 allele. Genotyping performed on samples from the proband's parents indicated that the father had the same FY genotype as the fetus. Flow cytometry, which has been previously demonstrated as a useful method to study antigen strength on cells, was used to determine if this new FY*01 allele was associated with reduced Fy(a) expression on the father's RBCs. Median fluorescence intensity of the father's RBCs (after incubation with anti-FY(a) and fluorescein-labeled anti-IgG) was similar to known FY*01 heterozygotes. and significantly weaker than known FY*01 homozygotes. In conclusion, the fetus and father both had one normal FY*01 allele and one new FY*01W.01, is associated with weakened expression of Fy(a) on RBCs.

Drug-induced immune hemolytic anemia: the last 30 years of changes.

Drug-induced immune hemolytic anemia (DIIHA) is a rare condition that occurs primarily as a result of drug-induced antibodies, either drug-dependent or drug-independent. Drug- dependent antibodies can be detected by testing drug-treated red blood cells (RBCs) or untreated RBCs in the presence of a solution of drug. Drug-independent antibodies react with untreated RBCs (no drug added) and cannot be distinguished from warm autoantibodies. Many changes have occurred during the last 30 years, such as which drugs most commonly cause DIIHA, the optimal testing methods for identifying them, and the theories behind the mechanisms by which they react. This article reviews the major changes in DIIHA since the early 1980s involving the immune complex mechanism, cephalosporins, nonimmunologic protein adsorption, and penicillins. Because serologic results associated with DIIHA can mimic those expected with autoimmune hemolytic anemia or hemolytic transfusion reactions, DIIHA may go undetected in some cases.

Drugs that have been shown to cause drug-induced immune hemolytic anemia or positive direct antiglobulin tests: some interesting findings since 2007.

This review updates new findings in drug-induced immune- hemolytic anemia (DIIHA) since the 2007 review in Immunohematology by these authors. Twelve additional drugs have been added to the three tables listing drugs associated with drug-dependent antibodies, drugs associated with drug-independent antibodies, and drugs associated with nonimmunologic protein adsorption. Other updated findings include (1) piperacillin is currently the most commonly encountered cause of DIIHA, (2) new data on blood group specificity of drug-dependent antibodies, (3) drug-dependent antibodies detected in healthy donors, (4) DIIHA associated with transplantation, and(5) DIIHA associated with chemotherapeutic drugs.

How we investigate drug-induced immune hemolytic anemia.

Drugs are a rare cause of immune hemolytic anemia, but an investigation for a drug antibody may be warranted if a patient has definitive evidence of immune hemolysis, other more common causes of hemolysis have been excluded, and there is a good temporal relationship between the administration of a drug and the hemolytic event. Drug antibodies are either drug-dependent (require drug to be in the test system) or drug-independent (reactive without drug present in the test). Drug-dependent antibodies are investigated by testing drug-treated red blood cells (RBCs) or by testing RBCs in the presence of a solution of drug. Drug-independent antibodies are serologically indistinct from idiopathic warm autoantibodies and cannot be defined or excluded by serologic testing. Nonimmunologic protein adsorption, caused by some drugs, is independent of antibody production but may also cause immune hemolytic anemia. Serologic methods for testing for drug antibodies are presented, and observations from more than 30 years of this laboratory's experience are discussed.

Serologic characteristics of ceftriaxone antibodies in 25 patients with drug-induced immune hemolytic anemia.

BACKGROUND: Ceftriaxone, a third-generation cephalosporin, is commonly used to prevent and treat infections. Since 1987, it has been the second most common cause of drug-induced immune hemolytic anemia (DIIHA) investigated in our laboratory. STUDY DESIGN AND METHODS: Samples from 79 patients (1987-2010), suspected of having DIIHA caused by ceftriaxone, were studied for the presence of ceftriaxone antibodies. Direct antiglobulin tests (DATs) and tests with ceftriaxone-treated red blood cells (RBCs) or untreated and enzyme-treated RBCs in the presence of ceftriaxone were performed. RESULTS: Twenty-five (32%) of the 79 patients had antibodies to ceftriaxone detected. Seventeen (68%) of the 25 patients were children; reactions in children were usually dramatic and severe. Nine (36%) of the 25 patients had fatal DIIHA. Nineteen of the 25 samples had DATs performed by our laboratory; 100% of samples were reactive with anti-C3 and 47% were reactive with anti-IgG. All 25 sera had ceftriaxone antibodies detected when testing untreated or ficin-treated RBCs in the presence of ceftriaxone (resulting in agglutination, hemolysis or sensitization of test RBCs). These antibodies were primarily IgM and reactivity was enhanced by testing ficin-treated RBCs. Sixteen (64%) of the 25 sera reacted with test RBCs when no ceftriaxone was added in vitro; this was most likely due to the transient presence of drug or drug-immune complexes in the patient's circulation at the time that the blood samples were drawn. CONCLUSION: Ceftriaxone antibodies can cause severe intravascular hemolysis. Complement can usually be detected on the patient's RBCs and IgM antibodies are usually detected in the patient's serum.

Immune hemolytic anemia due to cimetidine: the first example of a cimetidine antibody.

BACKGROUND: Although there have been a few reports of immune hemolytic anemia (IHA) thought to be due to cimetidine, none of them provided proof (e.g., serologic detection of anti-cimetidine and/or repeat of IHA upon drug rechallenge). One report used cimetidine as an example of how temporal associations of drug administration and hemolytic anemia are not proof of a cause-effect relationship. STUDY DESIGN AND METHODS: A 63-year-old cancer patient developed IHA on two occasions after receiving cimetidine (with and without chemotherapy). Serologic methods included testing cimetidine-treated red blood cells (RBCs) as well as testing untreated RBCs in the presence of cimetidine. RESULTS: The patient's direct antiglobulin test was positive (C3 only) and a serum antibody to cimetidine was detected by both testing methods. An eluate from the patient's RBCs was nonreactive. Cimetidine-treated RBCs were optimally prepared at room temperature and needed to be tested on the day of preparation. CONCLUSIONS: This is the first reported case of IHA due to a cimetidine antibody where a drug-dependent antibody was demonstrated. The patient had IHA after receiving cimetidine on two separate occasions.

Serologic findings in autoimmune hemolytic anemia associated with immunoglobulin M warm autoantibodies.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) associated with immunoglobulin M (IgM) warm autoantibodies is unusual but often severe, with more fatalities than other types of AIHA. Diagnosing this type of AIHA can be difficult because routine serologic data are not always informative. STUDY DESIGN AND METHODS: Forty-nine cases of IgM warm AIHA in 25 years were studied by serologic methods. RESULTS: Routine direct antiglobulin tests (DATs) detected red blood cell (RBC)-bound C3 in 90 percent of cases (65% had C3 but no immunoglobulin G [IgG] on their RBCs) and IgG in 24 percent. IgM was detected on 29 of 47 (62%) patients' RBCs; RBC-bound IgM was detected in 14 of 47 cases by a tube DAT method and in an additional 15 of 21 (71%) cases using fluorescein isothiocyanate anti-IgM and flow cytometry. Eighty-one percent of eluates from patients' RBCs reacted. Warm autoagglutinins were present in 94 percent of serum samples; untreated and enzyme-treated RBCs were hemolyzed at 37 degrees C by 13 and 65 percent of serum samples, respectively. Most agglutinins were optimally reactive at 30 to 37 degrees C. Patients' RBCs were spontaneously agglutinated in 78 percent of cases; washing with 37 degrees C saline or treating RBCs with dithiothreitol resolved this problem. Clear specificity of autoantibody was defined in 35 percent of serum samples. CONCLUSION: IgM warm AIHA can be confused with cold agglutinin syndrome and "mixed/combined"-type AIHA; a serologic workup by a specialist reference laboratory can help with the diagnosis.

Evaluation of potential immune response and in vivo survival of riboflavin-ultraviolet light-treated red blood cells in baboons.

BACKGROUND: Pathogen reduction methods have the potential to modify blood components, resulting in immunologic reactions or compromised blood components. This study evaluated the hypothesis that there is no immune response to riboflavin-and-ultraviolet [UV]-light-treated red blood cells (RBCs), as observed by serology and by survival of RBCs in circulation. STUDY DESIGN AND METHODS: Three baboons were in each treatment group: 1) untreated (negative control), 2) quinacrine mustard (QM)-treated (positive control), and 3) riboflavin-and-UV light-treated (test group) RBCs. In the immunization phase, autologous test or control RBCs were injected subcutaneously on Days 0, 21, 42, and 49. Plasma samples from these days were tested against test or control RBCs by flow cytometry and standard serology. On Day 56, autologous (51)Cr-labeled test or control RBCs were injected. Blood samples were taken over 21 days after injection to determine RBC survival (t(1/2)). RESULTS: Untreated and riboflavin-and-UV-light-treated RBCs showed no evidence of significant immunoglobulin G (IgG) binding after incubation with autologous plasma. RBC-bound IgG was detected on QM-treated RBCs after incubation with autologous plasma. This antibody was inhibited by QM, as demonstrated by a hapten inhibition study. t(1/2) values for the untreated and riboflavin-and-UV-light-treated RBCs were 7.3 +/- 0.8 and 7.5 +/- 1.7 days, respectively; the t(1/2) value for QM-treated RBCs was 2.3 +/- 2.9 days. CONCLUSION: Treatment with riboflavin and UV light did not render RBCs immunogenic. Positive controls indicated that immunization promoted an immune response. In the (51)Cr-labeled RBC survival phase of the study, riboflavin-and-UV-light-treated RBCs exhibited behavior similar to negative control RBCs. Detrimental immunologic or functional side effects were not observed.

Serological studies of piperacillin antibodies.

BACKGROUND: Penicillin-induced immune hemolytic anemia (IHA) is associated with immunoglobulin G antipenicillin detected by testing penicillin-coated red blood cells (RBCs). Antibodies to piperacillin, a semisynthetic penicillin, would be expected to react similarly; however, antipiperacillin can be detected by testing in the presence of the drug. Piperacillin is commonly used in combination with tazobactam, which causes nonimmunologic protein adsorption onto RBCs. In six cases of piperacillin-induced IHA, reactivity with piperacillin-coated RBCs was not similar to reactivity of antipenicillin with penicillin-coated RBCs. STUDY DESIGN AND METHODS: Antipiperacillin was tested against piperacillin-coated RBCs prepared using different pH buffers. Plasma from blood donors and sera/plasma from patients were tested with piperacillin-coated, penicillin-coated, and uncoated RBCs. Hapten inhibition studies were performed using different concentrations of piperacillin. Donors' plasma were tested in the presence of piperacillin; sera from patients with IHA were tested in the presence of tazobactam. RESULTS: Piperacillin required high pH for binding to RBCs. Agglutination of piperacillin-coated RBCs was observed in 91 percent of donors' and 49 percent of patients' plasma and was inhibited by piperacillin. In contrast to patients with IHA due to piperacillin, donors' plasma tested in the presence of piperacillin did not react. Tazobactam antibodies were not detected. CONCLUSION: A high percentage of donors' and patients' plasma contain an antibody to piperacillin or a chemically related structure detected by testing with piperacillin-coated RBCs. A diagnosis of piperacillin-induced IHA should not be made solely on the reactivity of a patient's plasma/serum with piperacillin- or piperacillin/tazobactam-coated RBCs; testing in the presence of piperacillin is more reliable.

Blood doping in athletes--detection of allogeneic blood transfusions by flow cytofluorometry.

Athletes may undergo blood transfusion to increase their red cell mass and the oxygen carrying capacity of their blood in order to confer a competitive advantage. Allogeneic transfusions are normally mismatched at one or more minor blood group antigens. The most sensitive and accurate method known to detect this form of blood doping is flow cytometry. Low percentages of antigen-positive and antigen-negative red blood cells (RBCs) can be quantitated using suitable specific alloantibodies and careful analysis. By testing blood samples taken at various times, a reduction in the percentage of a minor population of RBCs will indicate transfusion has occurred.

A 13-year-old girl with cold agglutinin syndrome caused by anti-i.

SUMMARY: A 13-year-old girl with cold agglutinin syndrome caused by anti-i was serologically positive for Epstein-Barr virus. The anti-i had a high titer at 4 degrees C and high thermal amplitude (reacting up to 37 degrees C with both cord i RBCs and the patient's autologous RBCs). The patient's hemoglobin dropped to 48 g/L. The age of the patient, the severity of the hemolysis, and the antibody specificity were unusual features of cold agglutinin syndrome. Transfusions with adult (I) red blood cells were effective.

Hemolytic disease of the fetus and newborn due to anti-Ge3: combined antibody-dependent hemolysis and erythroid precursor cell growth inhibition.

The Gerbich (Ge) antigens are a collection of high-incidence antigens carried on the red blood cell membrane glycoproteins, glycophorins C and D. Antibodies against these antigens are uncommon, and there have been only rare case reports of hemolytic disease of the fetus and newborn due to anti-Ge. In this case report, we present a neonate with severe anemia and hyperbilirubinemia due to anti-Ge3. Routine and special laboratory studies undertaken in this case suggested two mechanisms for the patient's hemolysis and persistent anemia. Antibody-dependent hemolysis was associated with early-onset hyperbilirubinemia, anemia, and a mild reticulocytosis, and inhibition of erythroid progenitor cell growth was associated with late anemia and normal bilirubin and reticulocyte values. Though rare, anti-Ge3 can be a dangerous antibody in pregnancy. Affected neonates may require intensive initial therapy and close follow-up for at least several weeks after delivery.

The changing spectrum of drug-induced immune hemolytic anemia.

Drug-induced immune hemolytic anemia (DIIHA) occurs rarely. To date, about 100 drugs have been implicated in causing DIIHA and/or a positive direct antiglobulin test (DAT). The most common drugs associated with DIIHA in the 1970s were methyldopa and penicillin; currently, they are cefotetan and ceftriaxone. Drug antibodies fall into two types: drug-independent ("autoantibodies") and drug-dependent ("penicillin type" or "immune complex type"); some patients have combinations of these antibodies. Some drugs cause nonimmunologic protein adsorption onto drug-treated red blood cells (RBCs). This is known to be the cause of positive indirect antiglobulin tests and is suspected to be a cause of positive DATs. This mechanism may be associated with hemolytic anemia. Twelve cephalosporins have been reported to cause DIIHA; five (primarily cefotetan and ceftriaxone) have been associated with fatalites. Patients with DIIHA due to cefotetan may only have received one dose of the drug prophylactically with surgery. Antibodies to cefotetan react to very high titers against drug-treated RBCs (and at lower titers against untreated RBCs without and/or with drug present). Patients with ceftriaxone-induced DIIHA have received the drug previously; reactions in children often occur minutes after ceftriaxone administration. Antibodies to ceftriaxone are only of the "immune complex type."

Cross-reactivity of cefotetan and ceftriaxone antibodies, associated with hemolytic anemia, with other: cephalosporins and penicillin.

Most drug-induced immune hemolytic anemias since the late 1980s have been caused by the second- and third-generation cephalosporins, cefotetan and ceftriaxone, respectively. Cross-reactivity of cefotetan and ceftriaxone antibodies with other cephalosporins or penicillin has been studied only minimally. We tested 7 serum samples previously identified to contain cefotetan antibodies and one serum sample previously identified to contain ceftriaxone antibodies against 9 other cephalosporins, penicillin, and 7-aminocephalosporanic acid in the presence of RBCs and also used hapten inhibition to indicate cross-reactivity. Serum samples containing cefotetan antibodies showed some cross-reactivity with cephalothin and cefoxitin (and to a much lesser extent with penicillin and ceftazidime). The ceftriaxone antibodies showed very weak cross-reactivity with cefotaxime, cefamandole, and cefoperazone. There was very little cross-reactivity between cefotetan antibodies and the drugs tested in the present study. We have no data to determine whether the in vitro data relate to in vivo reactivity.

A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination.

Flow cytometry operators often apply familiar white blood cell (WBC) methods when studying red blood cell (RBC) antigens and antibodies. Some WBC methods are not appropriate for RBCs, as the analysis of RBCs requires special considerations, for example, avoidance of agglutination. One hundred seventy-six published articles from 88 groups studying RBC interactions were reviewed. Three fourths of groups used at least one unnecessary WBC procedure for RBCs, and about one fourth did not use any method to prevent/disperse RBC agglutination. Flow cytometric studies were performed to determine the effect of RBC agglutination on results and compare different methods of preventing and/or dispersing agglutination. The presence of RBC agglutinates have been shown to be affected by the type of pipette tip used for mixing RBC suspensions, the number of antigen sites/RBC, the type and concentration of primary antibody, and the type of secondary antibody. For quantitation methods, for example, fetal maternal hemorrhage, the presence of agglutinates have been shown to adversely affect results (fewer fetal D+ RBCs detected).