Profound autoimmune hemolysis and Evans syndrome in two asplenic patients with babesiosis.

BACKGROUND: Evans syndrome (ES) is characterized by the simultaneous or sequential presence of multiple autoimmune cytopenias. It is often secondary to rheumatologic disorders or lymphoid malignancies, but has not previously been associated with babesiosis. Here we present two cases of severe cytopenias in asplenic patients precipitated by active babesiosis. CASE REPORT: The first patient had a history of Hodgkin's lymphoma in remission and autoimmune hemolytic anemia (AIHA) treated by splenectomy 12 years prior who presented with severe AIHA and thrombocytopenia after Babesia infection. The second patient had a history of ES requiring splenectomy, which relapsed after Babesia infection. RESULTS: The complex presentation and medical histories of both patients made the diagnosis challenging. Both patients' cytopenias responded to therapy, although the use of immunosuppressive agents in patients with active hematologic infections was challenging and required a multidisciplinary approach. CONCLUSION: These two cases illustrate the possibility of babesiosis to not only reactivate ES in asplenic patients, but also precipitate increased levels of immune deregulation, potentially provoking ES, a phenomenon not previously reported.

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.

Anti-Ge3 causes late-onset hemolytic disease of the newborn: the fourth case in three Hispanic families.

BACKGROUND: The Gerbich (Ge) blood group system consists of 11 antigens carried on red blood cell (RBC) membrane glycophorins C and D; of these, Ge:3 antigen is of high prevalence, and the anti-Ge3 is found to be clinically significant. CASE REPORT: A 34-week neonate born to a Hispanic mother with anti-Ge3 developed late-onset hemolysis with hyperbilirubinemia and was successfully treated with transfusions from her mother. Relevant clinical findings and laboratory results for this case are summarized and compared to three other previously reported cases; all babies were born from a mother of Hispanic ethnicity. CONCLUSION: Hemolytic disease of the fetus and new born associated with anti-Ge3 is rare but should be considered when working up a broadly reactive RBC antibody screen in women of Hispanic ethnicity. Early identification of pregnant women with anti-Ge3 is recommended for prenatal transfusion planning and close monitoring of the newborn infant for evidence of late-onset anemia.

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.

Long-term increases in lymphocytes and platelets in human T-lymphotropic virus type II infection.

Human T-lymphotropic viruses types I and II (HTLV-I and HTLV-II) cause chronic infections of T lymphocytes that may lead to leukemia and myelopathy. However, their long-term effects on blood counts and hematopoiesis are poorly understood. We followed 151 HTLV-I-seropositive, 387 HTLV-II-seropositive, and 799 HTLV-seronegative former blood donors from 5 U.S. blood centers for a median of 14.0 years. Complete blood counts were performed every 2 years. Multivariable repeated measures analyses were conducted to evaluate the independent effect of HTLV infection and potential confounders on 9 hematologic measurements. Participants with HTLV-II had significant (P < .05) increases in their adjusted lymphocyte counts (+126 cells/mm(3); approximately +7%), hemoglobin (+2 g/L [+0.2 g/dL]) and mean corpuscular volume (MCV; 1.0 fL) compared with seronegative participants. Participants with HTLV-I and HTLV-II had higher adjusted platelet counts (+16 544 and +21 657 cells/mm(3); P < .05) than seronegatives. Among all participants, time led to decreases in platelet count and lymphocyte counts, and to increases in MCV and monocytes. Sex, race, smoking, and alcohol consumption all had significant effects on blood counts. The HTLV-II effect on lymphocytes is novel and may be related to viral transactivation or immune response. HTLV-I and HTLV-II associations with higher platelet counts suggest viral effects on hematopoietic growth factors or cytokines.

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.

A severe case of cefoxitin-induced immune hemolytic anemia.

Drug-induced immune hemolytic anemia is a rare but underdiagnosed and potentially fatal condition. We report a case of severe hemolytic anemia induced by cefoxitin in a 45-year-old woman admitted with menometrorrhagia. Hemoglobin levels reached a nadir of 4.7 g/dl approximately 72 h after cefoxitin initiation, and hemolysis resolved when cefoxitin was discontinued and prednisone 1 mg/kg was initiated. A transfusion reaction workup revealed no abnormalities. Direct antiglobulin testing was weakly positive with anti-C3. The patient's plasma and RBC eluate reacted with cefoxitin-treated RBCs but not with untreated RBCs in the presence or absence of cefoxitin.

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.

Positive direct and indirect antiglobulin tests associated with oxaliplatin can be due to drug antibody and/or drug-induced nonimmunologic protein adsorption.

BACKGROUND: Two patients were suspected of having immune hemolytic anemia (IHA) due to oxaliplatin. A related drug, cisplatin, is known to cause nonimmunologic protein adsorption (NIPA). Studies were performed to determine the presence of oxaliplatin-dependent antibodies in addition to oxaliplatin-induced NIPA. STUDY DESIGN AND METHODS: Sera and eluates from the two patients were tested against red blood cells (RBCs) treated with oxaliplatin, cisplatin, and carboplatin (another platinum drug). Sera were also tested against untreated RBCs in the presence of the same drugs. Testing with pooled normal sera and anti-human albumin was used to demonstrate the presence of NIPA. Oxaliplatin-treated RBCs sensitized with the patients' sera and pooled normal sera were tested by a monocyte monolayer assay (MMA) to determine potential clinical significance. RESULTS: Both patients had high-titer antibodies to oxaliplatin in their sera that reacted with oxaliplatin-treated RBCs and with untreated RBCs in the presence of oxaliplatin. RBCs treated with oxaliplatin, cisplatin, and carboplatin all demonstrated NIPA (pooled normal sera and anti-human albumin were reactive to low titers). NIPA was also detected in tests with untreated RBCs in the presence of oxaliplatin and cisplatin. Lower-titer reactivity of both patients' sera with cisplatin may have been due to NIPA and/or cross-reactivity of anti-oxaliplatin with cisplatin. MMAs were weakly positive due to NIPA and more strongly positive due to oxaliplatin antibodies. CONCLUSION: Two patients with IHA were demonstrated to have oxaliplatin-dependent antibodies. Oxaliplatin was also shown to cause NIPA. The drug-dependent antibody and/or the drug-induced NIPA could have contributed to the patients' hemolytic anemia.

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.

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.

Sir John Dacie, MD, FRCP, FRCPath, FRS (1912-2005).

Sir John Dacie would be accepted by most of us as the father of modern hematology in the UK, and many would argue that this could be extended to other countries because so many people of other nationalities were trained in his laboratory in London. In his specialty of hemolytic anemia, he dominated the field, and his book (5 volumes) set the standard for investigators in this area. Luckily, he lived for 92 years (1912-2005) and kept a sharp mind up to his death, continuing to contribute to the medical literature into his 80s.

Immune hemolytic anemia associated with negative routine serology.

Immune hemolytic anemia can occur in patients who have no antibodies detectable by routine procedures (direct [DAT] and indirect [IAT] antiglobulin tests). DAT-negative autoimmune hemolytic anemias (AIHAs) represent 5% to 10% of all AIHAs. Three causes have been identified: (1) small numbers of red blood cell (RBC)-bound IgG molecules below the threshold of the DAT; (2) IgA and IgM autoantibodies; and (3) low-affinity autoantibodies. Antibody-independent cytotoxic events caused by natural killer (NK) cells have also been implicated. DATs are sometimes found to be positive when tested by reference laboratories, due to poor technique in reading antiglobulin tests in hospital laboratories. Hemolytic transfusion reactions also can occur when no alloantibodies are detectable by routine procedures. In some cases antibodies can be detected by special serologic procedures (such as the Polybrene test); in other instances phenotypically matched RBCs survive well and a specific antigen can be shown to be involved, suggesting a specificity (like anti-C) that is undetectable by any technique. Antibodies other than to blood group antigens, such as human leukocyte antigen (HLA) antibodies, may sometimes be involved.

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."