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Background: Autoimmune hemolytic anemia (AIHA) caused by warm IgG antibodies is usually characterized by IgG-mediated extravascular hemolysis and anemia that develops slowly. We report a case of AIHA presenting with sudden massive intravascular hemolysis and disseminated intravascular coagulation (DIC), similar to that seen following an ABO incompatible transfusion reaction. Case Report: A 71-year-old female had a 4-year history of immune thrombocytopenic purpura requiring splenectomy 1 ½ years ago. Six weeks prior to admission, in association with a viral infection, her platelet (plt) count fell from 350,000/mm3 to 150,000/mm3 over 2 weeks. She was treated with oral prednisone, and one week prior to admission her platelet count was 200,000/mm3. Two days before admission she noted reddish urine. Laboratory studies at her doctor’s office showed hemoglobin (Hb) 9 g/dL (had been 13 g/dL 1 week earlier) and bilirubin (bili) 9 mg/dL. On the day of admission, in the emergency department, she complained of generalized weakness, jaundice, and near-syncope. She became increasingly agitated and dyspneic and was intubated. At that time laboratory studies showed Hb 4.5 g/dL, LDH >2,500 U/L, haptoglobin 7 mg/dL, total bili 13.0 mg/dL, indirect bili 11.8 mg/dL, D-dimers >10,000 ng/dL, plts 139,000/mm3, prothrombin time 18.3 seconds, fibrinogen 166 mg/dL, fibrin monomers were positive. She was admitted to the medical intensive care unit with a diagnosis of DIC and hemolysis. The patient’s RBCs were coated with IgG (4+) and C3 (3+); IgM and IgA were not detected. Her plasma and an eluate contained an antiglobulin test reactive autoantibody with anti-Ena specificity (anti-Pr and -Wrb were excluded), an antibody directed at epitopes on glycophorin A. No alloantibodies were detected in the patient’s plasma. The patient was treated with IV steroids and received multiple RBC transfusions over the first few hospital days. Her Hb stabilized at 10 g/dL. The day after admission, she became oliguric and required dialysis throughout her hospital course. Over the next several days, additional signs and symptoms of DIC were noted: ischemic digits and toes, elevated Troponin I, partial thromboplastin time, D-dimers and decreased plt count. She was placed on intravenous heparin. Gastrointestinal bleeding developed and heparin was discontinued on hospital day 9. One week later, the onset of abdominal pain necessitated exploratory laparotomy which revealed ischemic bowel with perforation; vessels showed organized thrombi with recanalization. She remained critically ill and died on hospital day 25. Conclusion: Complement-mediated intravascular hemolysis in a patient with AIHA due to anti-Ena was associated with renal failure and DIC leading to death of the patient. All of these, even separately, are unusual in warm AIHA. We know of no reports where all 5 events (intravascular lysis, anti-Ena, renal failure, DIC, and death) occurred in one patient.

Background: Rapid clearance of poly(ethylene glycol)-asparaginase (PEG-asparaginase) has been reported for up to one third of patients treated for acute lymphoblastic leukemia, potentially rendering their treatment ineffective [Hempel G, Lanvers-Kaminsky C, Mueller, HJ, Würtwein G, Boos J. Blood. 2004;11:751A]. Poly(ethylene glycol) is an inert, biocompatible synthetic polymer and has long been claimed to be non-immunogenic; several PEG-conjugated drugs are FDA approved and many other PEGylated agents are in development. However, we have previously reported a 25% occurrence of an antibody against PEG (anti-PEG) in healthy blood donors, which suggests that PEG is an immunogenic agent. [Garratty G. Trans Med Rev. 2004;18:245–56]. Objective: To determine whether an anti-PEG could account for the rapid clearance of PEG-asparaginase. Methods: This study re-analyzed stored serum samples from pediatric patients who were enrolled in the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster 2000 studies. Samples were selected to include 15 subjects who showed undetectable asparaginase activity after receiving PEG-asparaginase (Oncaspar®) and 13 with normal sustained levels of asparaginase activity after treatment with PEG-asparaginase. Sixteen subjects treated with unmodified asparaginase (Medac®) were also included, 8 with negligible asparaginase activity. Sera were tested for the presence of an anti-PEG using two techniques: 1) Serology, by agglutination of PEG-coated red blood cells; 2) Flow cytometry, by analysis of 10 μm PEG beads (Tentagel®-OH) pre-incubated with sera and stained for bound immunoglobulins with fluorescein-anti-human IgG and R-phycoerythrin-anti-human IgM. Testing for anti-PEG was performed in a blinded manner. Results: For the PEG-asparaginase treated patients, of the 15 who showed undetectable asparaginase activity, 9 tested positive for anti-PEG by serology and 13 tested positive for anti-PEG by flow cytometry. All PEG-asparaginase treated patients with normal sustained asparaginase activity tested negative for anti-PEG. Four sera yielded somewhat unusual results: One weak IgM anti-PEG positive sample showed measurable asparaginase activity and three anti-PEG negative patients had low asparaginase activity, two of which were positive for anti-asparaginase. No relationship was observed between the presence of anti-PEG and serum asparaginase activity for the patients treated with unmodified asparaginase (Medac®). Conclusions: The presence of anti-PEG was very closely associated with rapid clearance of PEG-asparaginase. Screening and monitoring for anti-PEG would allow for identification of a subset of patients for whom a modified dosing strategy or use of a non-PEGylated drug would be appropriate.

Background: S-303 (an acridine compound) treatment of RBCs inactivates a broad spectrum of pathogens. Two randomized, controlled Phase III trials of OSRBC to support patients (pts) undergoing cardiovascular surgery (CVS) or with hemoglobinopathies (HB) were in progress when antibodies (Ab) to OSRBC were detected. The specificity of these Ab was determined to be due to residual RBC-bound acridine. The S-303 treatment process was modified in an attempt to reduce immunoreactivity and immunogenicity. MSRBC show reduced RBC-bound S-303 and no reactivity with anti-S-303 Ab. Methods: Retrospective central laboratory testing was performed on all available trial pt serum samples (multiple time points for each pt). Crossmatch (CM) testing (PEG CM) against a minimum of 3 OSRBC units and the paired pre-treatment RBC segment was performed for each sample. CM was defined positive when reactive with any OSRBC, and consistent with anti-S-303 Ab when reactive with all OSRBC, but none of the paired pre-treatment RBC. CM-positive sera were tested for S-303 specificity with a CM inhibition assay using S-303 analogues and also tested for CM reactivity against MSRBC with a gel card CM test. All available baseline sera from trial pts, as well as plasma from 200 healthy blood donors, were also tested by gel card CM against OSRBC and MSRBC to determine prevalence of Ab reactivity to SRBC in absence of prior SRBC exposure. Results: Sera from 8 of 174 (4.6%) pts from both trials showed some CM reactivity with OSRBC. 4 were positive with all ORBC tested; and 4 were inconsistently positive, reactive with some but not all OSRBC. For the 4 pts (2.3%) with consistently positive CM, 3 had OSRBC exposure (HB trial) and 1 had only Control RBC exposure (CVS trial); 3 had positive CM with S-303 specificity. None of these 4 pt sera was CM positive with MSRBC. Only 1 of the other 4 pts with inconsistently positive CM tests had a CM test specific for SRBC (a Control CVS pt); sera from this pt were not reactive with MSRBC. 4 (1.8%) baseline sera from 220 trial pts (205 CVS and 15 HB) were CM-positive with OSRBC; only 2 (0.9%) were CM-positive with all units tested; none was positive with MSRBC. 3 of 200 (1.5%) healthy blood donor plasma were CM-positive with OSRBC; only 2 were positive with all units tested; none was positive with MSRBC. Conclusions: OSRBC were immunogenic in HB pts with prior chronic RBC exposure. Inconsistently positive CM to OSRBC without antigenic specificity for S-303 were detected with some pt sera; these results are under further investigation. Ab to OSRBC was detectable in sera of pts never exposed to S-303 treated RBC. Approximately 1% of pts and healthy blood donors had Ab that reacted with OSRBC. However, no sera with either inconsistent or confirmed CM reactivity to OSRBC were reactive with MSRBC. The MSRBC process eliminated CM reactivity to RBC treated with S-303 and offers the potential for pathogen inactivation treatment of RBC without immunologic reactivity.

Background: Paroxysmal cold hemaglobinuria (PCH) is caused by an IgG autoantibody which behaves as a biphasic hemolysin, attaching to RBCs at cold temperatures and activating complement at warmer temperatures, leading to hemolysis. This antibody, known as the Donath-Landsteiner antibody (DL-A), frequently shows specificity for the P-antigen. PCH was historically associated with syphilis infection. More recently, the DL-A has been found primarily in children with acquired autoimmune hemolytic anemia (AIHA) following a viral illness. In adults, PCH is rare and may occur as an idiopathic disease or in association with a lymphoproliferative disorder. Cases in children usually resolve spontaneously, whereas the adult form can be chronic and pose a therapeutic challenge, since treatment with steroids and splenectomy may be ineffective. Recently rituximab has been demonstrated to be a useful agent in treating AIHA that is resistant to conventional therapies. Case Report: A 64-year-old woman presented to another hospital with three months of progressive weakness. She was found to be severely anemic. Gastrointestinal blood loss was ruled out. Extensive work up was obtained with CT imaging and bone marrow biopsy, which showed no evidence of malignancy. A hemolytic process was identified and she was placed on oral prednisone 60mg daily. The patient then presented to Cedars-Sinai Medical Center three months later with recurrent fatigue and a hemoglobin concentration (Hb) of 6.6 g/dL. Lab values revealed an elevated reticulocyte count (7.9%), WBC 27.6, total bilirubin 3.5 mg/dL, indirect fraction 3.4 mg/dL, elevated LDH 445 U/L, absent haptoglobin, and microspherocytes on peripheral blood smear. The Direct Antiglobulin (Coombs) Test (DAT) was positive with an anti-complement reagent and negative with an anti-IgG reagent, leading to the suspicion of a DL-A or cold agglutinin. Cold agglutinin titer was normal. A Donath-Landsteiner test was positive, confirming the diagnosis of PCH. Steroids were rapidly tapered and she was given rituximab 375 mg/m2. Her Hb increased and evidence of hemolysis ceased. The patient received 3 additional doses of rituximab weekly. Her Hb recovered to normal. The patient did well for 9 months until she presented again with acute hemolysis (Hb 8.8 g/dL.) The DAT was again positive with an anti-complement reagent and negative with an anti-IgG reagent. She was given a single dose of rituximab with cessation of hemolysis. She received another 3 doses, which resulted in stabilization of her Hb. She remains well at 6 months follow-up. Discussion: The most frequent form of AIHA is due to a warm, IgG antibody and is commonly responsive to steroids or splenectomy, whereas in cold agglutinin disease, caused by an IgM antibody these therapies are usually ineffective. The use of rituximab has been reported as a useful treatment for both warm and cold AIHA refractory to conventional therapy. This is the first case report to our knowledge of a patient with adult PCH refractory to steroids successfully treated with rituximab. This patient responded dramatically to rituximab on two separate occasions, and has remained in remission since the second cycle after treatment with this single agent. Rituximab may represent an effective therapy for adult patients with chronic PCH.

Background : S-303 treatment (200 μM S-303 and 2 mM glutathione) has been shown to inactivate viruses, bacteria, protozoa and leukocytes in RBC concentrates. S-303-treated RBCs (SRBC) have comparable in vitro parameters to untreated conventional RBCs (CRBC) over 42 days (d) of storage. Three Phase I trials in healthy human subjects showed recovery and life span of 35 d old autologous S-303 RBC were comparable to that for CRBC without detectable antibody (Ab) formation. During the course of a Phase III trial for patients (pts) in chronic transfusion (txn) programs receiving repeated SRBC txn, 2 pts developed asymptomatic anti-SRBC Ab, leading to trial termination. Methods : A randomized, controlled, double-blinded, crossover design, non-inferiority trial of SRBC in pts with thalassemia (thal) or sickle cell anemia (SSA) was conducted. Fifty pts were to receive a course (≥5 months or ≥6 txn) of SRBC or CRBC with crossover to the other arm. The frequency and number of RBC units per txn and target hemoglobin (Hb) were at physician discretion. The primary endpoint was Hb txed per kg body weight per d. Secondary endpoints were: time between txn, pre-txn Hb, number of RBC units txed, Ab formation, and adverse events (AEs). Routine DAT and IAT with RBC panels, and cross match with IAT to SRBC (and to the pre-S-303 treatment segment if positive to SRBC), were performed at trial sites prior to each txn. Following detection of positive cross match to SRBC in 2 pts, all pre-txn samples from all pts were tested by polyethylene glycol (PEG) SRBC cross match with IAT at a reference lab. Post study termination, pts had monthly follow-up (for up to 6 months) for AEs and repeated SRBC PEG cross match IAT. Results : The trial was terminated after 26 pts received ≥1 txn when 2 asymptomatic pts in the SRBC arm developed positive (+) pre-txn IAT cross matches with SRBC but not the same RBC unit pre-S-303 treatment (SRBC Ab). No pt completed the trial; 69% started the 1st period only and 31% crossed over to the 2nd period. 17 pts had ≥1 SRBC txn. Neither pt with (+) cross matches had clinical evidence of significantly reduced RBC survival. Hapten inhibition assays showed inhibition by S-303 derivatives but not glutathione used in the treatment process; a monocyte monolayer RBC phagocytic assay was negative with pt sera, suggesting these Ab were not likely clinically significant. Six-month follow-up of all txed pts did not reveal additional SRBC Ab or AEs. No Ab to SRBC were detected in a companion Phase III trial of SRBC txn for pts undergoing cardiac surgery supported with SRBC txn for up to 7 d. Conclusions : Two of 17 pts with exposure to SRBC in chronic txn programs developed Ab to SRBC after txn with SRBC. These Ab did not appear to result in decreased RBC survival (e.g., hemolysis), AEs, autoantibody formation, or Ab formation to RBC antigens. None of 74 pts txed with SRBC for up to 7 d to treat anemia of cardiac surgery developed SRBC Ab. A modified S-303 treatment process to reduce immunogenicity has been developed. Patients With Antibodies to S-303 RBC Patient 1 Patient 2 Age (yr) 3 13 Sex F M Diagnosis Thal SSA Time on study (d) 139 58 AEs Splenomegaly Pt. blood type AB+ A+ DAT/IAT (allo-Ab panel RBC) −/− −/− No. SRBC txn/units before (+) crossmatch 5/10 1/1 IAT reaction (LISS/PEG) against SRBC 2+/3+ 3+/2+ Titer of (+) sera against SRBC 2 8

We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.

A patient with idiopathic paroxysmal cold hemoglobinuria had severe hemolytic anemia despite minimal exposure to cold. The patient’s autoantibody demonstrated all the characteristic features of Donath-Landsteiner antibodies, except that strong hemolysis occurred with simple cooling (monophasic hemolysis), as well as with traditional cold-warm incubation (biphasic hemolysis). Monophasic hemolysis was caused by a Donath-Landsteiner antibody of exceptionally high thermal range, which caused red cell sensitization and hemolysis up to 32°C. The patient’s severe hemolytic anemia was probably a result of the high thermal range of the autoantibody, which would allow intravascular hemolysis with even minimal exposure to cold. Corticosteroids had a beneficial effect in controlling hemolysis; however, strict environmental control, with vigorous prophylaxis against even minimal cold, was the most important part of the therapeutic program.