Differential red blood cell age fractionation and Band 3 phosphorylation distinguish two different subtypes of warm autoimmune hemolytic anemia.

Warm autoimmune hemolytic anemia (wAIHA) is a blood disorder characterized by the increased destruction of autologous red blood cells (RBCs) due to the presence of opsonizing pathogenic autoantibodies. Preliminary reports published more than three decades ago proposed the presence of two wAIHA subtypes: Type I, in which autoantibodies preferentially recognize the oldest, most dense RBCs; and Type II, characterized by autoantibodies that show no preference. STUDY DESIGN AND METHODS: We evaluated patients having wAIHA for Type I and II subtype using discontinuous Percoll gradient age fractionation and direct antiglobulin test (DAT). We performed Western immunoblotting and mass spectrometry to show autoantibody specificity for Band 3. We investigated Band 3 tyrosine phosphorylation in different Percoll fractions to determine aging associated with oxidative stress. RESULTS: We confirm the existence of two subtypes of wAIHA, Type I and Type II, and that autoantibodies recognize Band 3. Type I patients were characterized by five Percoll fractions, with a DAT showing IgG opsonization F1 < F5 and elevated Band 3 phosphorylation compared to healthy controls (HCs). In contrast, Type II wAIHA patients were characterized by three to four Percoll fractions, where the DAT IgG opsonization shows F1 ≥ F3/4 and Band 3 phosphorylation was absent or significantly decreased compared to HC. CONCLUSIONS: Type I patients have increased Band 3 tyrosine phosphorylation that may represent accelerated aging of their RBCs resulting in exacerbation of a pathologic form of RBC senescence. Type II patients show decreased Band 3 tyrosine phosphorylation and lack the oldest, most dense RBCs suggesting premature RBC clearance and a more severe wAIHA.

Novel mutations in KLF1 encoding the In(Lu) phenotype reflect a diversity of clinical presentations.

BACKGROUND: Mutation in the KLF1 gene is the cause of the In(Lu) (Inhibitor of Lutheran) Lu(a-b-) phenotype and more than 60 alleles have been associated with this phenotype. Here we describe findings from investigation of seven cases: six presenting with a Lu(a-b-) phenotype including the historical index case and one referred from a patient with chronic anemia. STUDY DESIGN AND METHODS: Serologic testing was by standard methods. DNA testing included amplification and sequencing of KLF1 and LU coding regions. A StuI polymerase chain reaction-restriction fragment length polymorphism was designed to target c.304T>C in KLF1. RESULTS: Five different KLF1 alleles were identified. Three are new: KLF1*90A (p.Trp30Ter), KLF*911A (p.Thr304Lys), and KLF1*304C,318G (p. Ser102Pro, Tyr106Ter) present in two unrelated individuals. Two, including the index case, had c.954dupG (p.Arg319Glufs*34), that is, KLF1*BGM06. The child with unexplained anemia had c.973G>A (p.Glu325Lys), associated with congenital dyserythropoietic anemia. The common c.304T>C was found in two of the seven samples investigated and in 60 of 100 blood donors. CONCLUSION: Mutations in KLF1 are pleiotropic and although most are benign, others are associated with hematologic abnormalities. We report three new KLF1 alleles associated with benign In(Lu) and document both the molecular basis of the original In(Lu) phenotype using a frozen sample stored for more than 50 years and the cause of unexplained anemia in a child. We also confirm previous observations that c.304C (p.102Pro) is not, by itself, associated with an In(Lu) phenotype in donors self-identified as U.S. minorities.

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.

Drug-induced immune hemolytic anemia associated with anti-carboplatin and the first example of anti-paclitaxel.

BACKGROUND: Combined chemotherapy with carboplatin and paclitaxel is first-line treatment for lung and ovarian cancer. Drug-induced antibodies to carboplatin are rare but can cause severe, even fatal, hemolysis. Paclitaxel-induced immune hemolysis has not been reported. We describe a case of immune-mediated hemolysis associated with antibodies to carboplatin and paclitaxel in a woman with ovarian cancer who had received multiple chemotherapeutic agents over 7 years, including several courses of these two drugs. She required many transfusions. During a chemotherapy infusion the patient became hypotensive, was pale, and had rigors and red urine. The nadir hematocrit was 12.4%; peak bilirubin and lactate dehydrogenase were 16.3 mg/dL and 1188 units/L, respectively. STUDY DESIGN AND METHODS: Blood samples collected within hours after chemotherapy and 2 days later were tested for antibodies to carboplatin and paclitaxel. RESULTS: The direct antiglobulin test was positive with anti-IgG (3+) and anti-C3 (2+). The plasma collected shortly after chemotherapy agglutinated carboplatin-treated red blood cells (RBCs); untreated and paclitaxel-treated RBCs both reacted at the antiglobulin test most likely due to circulating carboplatin, paclitaxel, or both drugs. Serum collected 2 days later agglutinated (titer 2) and sensitized (titer 128) carboplatin-treated RBCs; untreated and paclitaxel-treated RBCs were nonreactive. An acid eluate reacted weakly in the presence of polyethylene glycol with carboplatin-treated RBCs. The serum reacted with untreated and enzyme-treated RBCs in the presence of soluble carboplatin and paclitaxel. CONCLUSION: Anti-carboplatin and the first example of anti-paclitaxel were detected in this patient's sample.

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.

Antibodies to oxaliplatin, a chemotherapeutic, are found in plasma of healthy blood donors.

BACKGROUND: Oxaliplatin is one of the platinum chemotherapeutics that includes cisplatin and carboplatin. Antibodies to all three drugs have caused immune hemolytic anemia (IHA). In an investigation of oxaliplatin-induced IHA, the negative plasma control agglutinated oxaliplatin-coated red blood cells (RBCs). Previous preparations of this control had not agglutinated oxaliplatin- or cisplatin-coated RBCs. STUDY DESIGN AND METHODS: Drug-coated RBCs, prepared by incubating 1/10th volume of RBCs with 1 mg/mL drug in phosphate-buffered saline for 1 hour at 37°C, were incubated with plasma from random blood donors and patients. Plasma was treated with dithiothreitol to determine the immunoglobulin class. Hapten inhibition was performed by incubating plasma with solutions of oxaliplatin or cisplatin. RESULTS: Nineteen of 121 (16%) donors' plasma samples agglutinated oxaliplatin-coated RBCs; 7 of 102 (7%) donors' plasma samples agglutinated cisplatin-coated RBCs. Two of 50 (4%) patients' samples agglutinated oxaliplatin-coated RBCs. The agglutinin was immunoglobulin M and inhibited by oxaliplatin and cisplatin. CONCLUSION: An agglutinin reactive with oxaliplatin-coated RBCs was found in 16% of donors' and 4% of patients' samples. Inhibition by oxaliplatin and cisplatin indicates the antibody may be directed to platinum. The presence of this antibody in healthy individuals may be related to the increasing environmental presence of platinum in air and soil as a byproduct of automobile catalytic converters and pharmaceuticals in our water and food chain. This antibody in individuals without IHA suggests that testing untreated and enzyme-treated RBCs in the presence of a solution of drug may be the best method to investigate IHA caused by drugs in the platinum family.

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.

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.

Fatal carboplatin-induced immune hemolytic anemia in a child with a brain tumor.

Drug-induced immune hemolytic anemia (DIIHA) is an uncommon side effect of pharmacologic intervention. A rare mediator of DIIHA, carboplatin is an agent used to treat many pediatric cancers. We describe here, the first case of fatal carboplatin induced DIIHA in a pediatric patient and a brief review of the literature. Our patient developed acute onset of multi-organ failure with evidence of complement activation, secondary to a drug induced red cell antibody. Early recognition of the systemic insult associated with carboplatin induced hemolytic anemia may allow for future affected patients to receive plasmapheresis, a potentially effective therapy.

Piperacillin-induced immune hemolytic anemia in an adult with cystic fibrosis.

We report a case of drug-induced immune hemolytic anemia (DIIHA) in an adult female with cystic fibrosis (CF), complicating routine treatment of a pulmonary exacerbation with intravenous piperacillin-tazobactam. Workup revealed a positive direct antiglobulin test (DAT) due to red blood cell (RBC)-bound IgG and C3 and piperacillin antibodies detectable in the patient's serum. The potential influence of CF transmembrane conductance regulator mutations on the severity of DIIHA is discussed. This report illustrates the importance of early identification of DIIHA, a rare complication of a commonly utilized medication in CF.

Weakening or loss of antibody reactivity after prewarm technique.

BACKGROUND: The prewarm (PW) technique is a popular approach to determine whether cold antibodies are reacting at 37 degrees C or to detect potentially clinically significant antibodies in the presence of cold autoantibodies. The PW technique has been criticized because of unexpected loss of antibody reactivity and inappropriate use of the method. STUDY DESIGN AND METHODS: Sera containing 94 antibodies were tested by routine and PW PBS-, LISS-, and PEG-IAT. LISS-IAT was also compared to PW PBS-IAT. Sera were also tested to determine the role of low-affinity antibodies in loss of reactivity by PW methods. RESULTS: PW PBS-IAT and PW LISS-IAT showed that 40 and 47 percent of antibodies were weakened, respectively, compared to LISS-IAT; reactivity for 14 percent of antibodies was completely lost by each PW method. By PW PBS-IAT, 34 percent of antibodies were weakened compared to PBS-IAT. PW PEG-IAT showed weakened reactivity by 56 percent of antibodies compared to PEG-IAT; reactivity of seven out of seven PEG-dependent antibodies was completely lost. Of 67 antibodies, 19 percent were defined as low affinity. Of 64 samples tested by the PW method and for low-affinity antibodies, only 6 of 30 that showed decreased reactivity by the PW method appeared to be due to low-affinity antibodies; only 6 of 12 samples that appeared to contain low-affinity antibodies also showed decreased reactivity by the PW method. CONCLUSION: Antibody reactivity of potentially clinically significant antibodies can be decreased or missed by PW methods. Antibody enhancement media does not ensure antibody detection by PW methods.