Clinical and Serological Characteristics of Cold Autoimmune Hemolytic Anemia with Concomitant Cold Agglutinin and Donath-Landsteiner Antibodies

Background The two major types of cold autoimmune hemolytic anemias are cold agglutinin disease (CAD) and paroxysmal cold hemoglobinuria (PCH). In CAD, the cold agglutinin is usually IgM with anti-I specificity. In PCH, the Donath-Landsteiner (DL) antibody is a cold reacting IgG with anti-P specificity. In this study, we describe the clinical and serological characteristics of patients with autoimmune hemolytic anemia positive for both cold agglutinin and DL antibodies. Methods On review of our immunohematology reference laboratory database, we identified patients over a 16-year period (January 2000- March 2016) with the following: i) age ≥18 years, ii) hemoglobin (Hb) <12g/dL, iii) positive direct antiglobulin test (DAT) with hemolysis (increased lactate dehydrogenase/low serum haptoglobin/ elevated indirect bilirubin), and iv) tested for both CAD and DL antibodies. We classified the patients into 3 cohorts. Cohort 1 included patients positive for both cold agglutinin (titer ≥1:64) and DL antibodies. Cohort 2 consisted of patients with DL antibody but no cold agglutinin (titer <1:64), while Cohort 3 was comprised of patients with cold agglutinin but no DL antibody. We evaluated the clinical response based on the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) criteria (Barcellini W, et al . Blood 2014) defined as i) complete (CR): hemoglobin ≥12 g/dL with normalization of at least one previously abnormal hemolytic marker; ii) partial (PR): hemoglobin 10-12 g/dL with hemolysis; or iii) none (NR): if any of the above criteria were not met. Results Seven patients had cold autoimmune hemolytic anemia with concomitant cold agglutinin and DL antibodies (Cohort 1). The clinical and serological characteristics are described in table 1. The median age at diagnosis was 68 years (range: 59-78) and the median hemoglobin at onset was 10.3 g/dL (range: 8.2-10.6). Six had red cell agglutination in the blood smear. Two patients had a recent history of infection (1 with Mycoplasma pneumoniae and 1 with upper respiratory tract infection). The median follow-up was 4.8 months (range: 1.0-166.4). Five patients received steroid/other immunosuppressants and two were managed conservatively. The clinical response to treatment was CR in 28.6% patients, PR in 42.9%, and NR in 14.3%. In comparison, Cohort 2 had a 20% CR, 20% PR and 40% NR, while Cohort 3 had a 50% CR, 25% PR and 25% NR, respectively. Conclusion Our study is the first series describing patients with cold autoimmune hemolytic anemia with concomitant cold agglutinin and DL antibodies. In terms of clinical response, patients with negative DL antibodies and cold agglutinin titers >1:64 had better response (50% CR, Cohort 3) to immunosuppressants compared to patients with positive DL antibody (20-30% CR, Cohorts 1 and 2). Large scale studies may be warranted to determine the treatment strategies among patients with concomitant DL and high cold agglutinin titers. Disclosures Kay: Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Tolero Corporation: Research Funding; Gilead: Research Funding. Winters: Fresenius Kabi USA: Consultancy; Eliaz Therapeutics Inc: Membership on an entity's Board of Directors or advisory committees; Sanofi Inc: Other: Moderated opinion leader's forum; Wiley Blackwell: Employment; Regional Health Inc: Consultancy; Mayo Clinic: Employment; Grifols International SA: Consultancy.

Life-Threatening Delayed Hemolytic Transfusion Reaction in a Patient with Sickle Cell Disease: Effective Treatment with Eculizumab Followed By Rituximab

Introduction: We report a life-threatening delayed hemolytic transfusion reaction (DHTR) with hyper-hemolysis syndrome (HHS) in a SCD patient triggered by an anti-IH autoantibody with alloantibody behavior. The intravascular hemolysis was successfully inhibited with Eculizumab therapy. The auto antibody was suppressed with rituximab treatment. Case Report: The patient is a 35 year-old African American female with SCD. She was given two red cell units ABO, Rh matched and negative for antigens to her known alloantibodies, before undergoing a laparoscopic cholecystectomy. She discharged with hemoglobin of 8.2 g/dL. Two weeks later she was admitted with severe fatigue, worsening jaundice, and total body pain. Physical examination was notable for scleral icterus, pale conjunctivae, and a well healed surgical scar on her abdomen. Hemoglobin level was 7.4 g/dL and reticulocyte count was reticulocyte count was 211x103/µl, lactate dehydrogenase (LDH) of 1174 IU/L (nl<225 IU/L ). Vital signs were normal. The next day, she spiked a fever of 39° C and her oxygen saturation dropped to 85-90% on 10 liters oxygen. Hemoglobin precipitously decreased to 3.8 g/dL, LDH greater than 5000 IU/L, total bilirubin 9.8 mg/dL, and haptoglobin less than 20 mg/dL. Serum creatinine (Cr) increased to 2.08 mg/dL from 0.77 mg/dL. Blood and urine cultures were negative. Chest x-raywas negative. A sample sent to the blood bank demonstrated brown cloudy plasma by visual inspection consistent with intravascular hemolysis. Cross matching of the patient’s plasma with segments from the two previously transfused units now showed incompatibility. The DAT was weakly positive for complement only. Testing performed at the American Red Cross (ARC) Immunohematology Reference Laboratory identified anti-IH cold agglutinin, reactive at room temperature and 37°C. Strong (4+) reactivity was observed with A2, B, and O red cells while weak (1+) reactivity was seen with autologous and A1 red cells. An acid elulate demonstrated strong reactivity with A2 and O red cells and no reactivity with autologous and A1 red cells. Initial 4°C cold agglutinin titer was 512. The high titer anti-IH antibody was of IgM subtype with reactivity with H antigen rich red cells (O and A2). Despite transfusion of five additional antigen negative RBC units, her hemoglobin further declined to 3.6 g/dL. Reticulocyte count also dropped to 36x103/µl and renal function further deteriorated (Cr = 2.08 mg/dL). Because of the aggressive complement mediated intravascular hemolysis, Eculizumab was given on a compassionate use protocol after approval from the USC Investigational Review Board. Meningococcal vaccination and prophylactic ciprofloxacin were given. Eculizumab dose was 1200 mg weekly for four weeks, followed by 1200 mg every 2 weeks starting on week 5. LDH dropped rapidly > 5000 IU/L to 1626 IU/L by day 12, 747 IU/L at day 22 and 467 IU/L by day 30. By day 7 of Eculizumab treatment, the patient’s hemoglobin stabilized at 5.4 g/dl without further need for transfusion with normalization of the serum creatinine (0.54 mg/dl). Immunosuppressive therapy with rituximab, 375mg/m2 was given weekly for 4 weeks to suppress the IH antibody. The cold agglutinin titer, initially noted to be 1:512, remained at 1:256 at 6 weeks but dropped to 1:4 by week 12. Discussion: We report a case of delayed hemolytic transfusion reaction with resultant hyperhemolysis triggered by an anti-IH autoantibody with allo-antibody behavior. The anti-IH was reactive at room temperature as well as 37oC, but only weakly reactive with autologous red cells treated with Rituximab to suppress the cold agglutinin and Eculizumab to block the complement mediated hemolysis After initiation of Eculizumab therapy our patient’s LDH levels decreased by 40% within the first week, 70% by day 14, and 90% at four weeks with stabilization of her hemoglobin without further transfusion. Conclusion: We have described the first reported case of HHS triggered by anti-IH formed as a result of red cell transfusion, successfully treated with Eculizumab and Rituximab. The rapid hematologic improvement due to Eculizumabv occurred well before the cold agglutinin titer dropped (7 days vs 84 days). Eculizumab may have utility in the treatment of other cases of severe DHTR, alone or, as in our patient, combined with rituximab. Disclosures Off Label Use: Eculizumab for inhibition of complement mediated hemolysis Rituximab for suppression of cold agglutinin. Liebman:Alexion: married to Dr. Ilene Weitz Other.

TNT009, a Classical Complement Pathway Specific Inhibitor, Prevents Complement Dependent Hemolysis Induced By Cold Agglutinin Disease Patient Autoantibodies

Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia in which autoantibodies bind to red blood cells (RBC) at temperatures below 37°C, resulting in activation of the classical complement pathway (CCP). CCP activation leads to hemolysis either intravascularly, by formation of the membrane attack complex, or extravascularly, when C3/C4 fragment deposition onto the RBC surface results in sequestration by the reticuloendothelial system. Here we describe the in vitro and in vivo activity of TNT003 and TNT009, inhibitors of a serine protease specific to the CCP, in pre-clinical models of CAD. TNT003 is a mouse monoclonal IgG2a antibody with sub-nanomolar affinity. TNT009 is the humanized form (IgG4) of TNT003 and retains affinity and specificity to its target. In vitro assays using IgM-sensitized sheep RBC and human or non-human primate (NHP) serum showed that TNT003 and TNT009 potently inhibited antibody-mediated hemolysis in a concentration dependent manner. Additionally, TNT003 and TNT009 inhibited CCP-mediated production of the anaphylatoxins C4a, C3a, and C5a. Flow cytometry analysis showed that both antibodies also prevented C3 fragment deposition on the RBC surface. Activity was proportional to the amount of serum used, and at 80% human or NHP serum, TNT003 completely inhibited hemolysis with an IC50 of ∼13 µg/mL. Using an ELISA-based assay, TNT003 inhibited C5b-9 deposition driven by the CCP but not by the alternative (CAP) or lectin (CLP) pathways. These data suggest that TNT003 and TNT009 are specific and potent inhibitors of the CCP. To demonstrate the utility of a CCP inhibitor in disease, we tested the ability of TNT003 and TNT009 to inhibit the CCP in ex vivo hemolysis assays using CAD patient autoantibodies. Type O- RBC were incubated in the presence of CAD plasma to sensitize the cells with autoantibody. RBC were then washed and 25% normal human serum (NHS) added as a source of complement. Thirteen of the seventeen CAD samples tested (76%) mediated C3 fragment deposition on the RBC surface as determined by flow cytometry. TNT003 significantly inhibited C3 fragment deposition by all patient samples that deposited complement (88 ± 2.6% inhibition, n = 13) with an average IC50 of 4.7 ± 0.4 µg/mL. One patient sample induced complement-dependent hemolysis of ∼50% of the RBC upon addition of NHS. In a concentration dependent manner, TNT003 and TNT009, but not control IgG, completely inhibited CAD autoantibody-mediated hemolysis (Fig. 1), as well as C4a, C3a and C5a generation. We further characterized each patient sample to determine cold agglutinin titer. We found that cold agglutinin titer correlated with the percent RBC staining positive for cell surface C3 fragments (R2 = 0.3566; p < .01; n = 17 samples; Fig. 2).Figure 1TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 1. TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 2Cold agglutinin titers correlate with C3 fragment deposition on RBCFigure 2. Cold agglutinin titers correlate with C3 fragment deposition on RBC Extravascular hemolysis of C3 fragment-coated RBC by liver macrophages is believed to be the primary mechanism of RBC destruction in CAD. We therefore tested the hypothesis that CAD patient plasma-induced C3 fragment deposition on RBC would promote phagocytosis by the monocytic cell line THP-1. We found that RBC sensitized in CAD plasma and exposed to NHS were engulfed in an FcgR-independent mechanism by THP-1 cells. RBC phagocytosis was significantly inhibited if NHS exposure occurred in the presence of TNT003 (100 µg/mL), but not a control IgG. The selective CCP inhibitory activity of TNT003 was evaluated in vivo in cynomolgus monkeys. TNT003 administered as a single IV injection at 30 mg/kg resulted in a Cmax of ∼330 µg/mL and detectable serum TNT003 thru ≥72 hours. Using an ELISA-based assay, we observed specific inhibition (≥95%) of the CCP for ≥72 hours. In contrast, CAP activity was modestly and transiently inhibited for 4 - 8 hours. At Cmax, endogenous C4a levels were reduced by >90% and returned to baseline levels by ≥96 hours. Serum samples containing TNT003 showed complete (100%) inhibition of hemolysis and C3 fragment deposition in vitro. CCP activity was completely restored to baseline after TNT003 concentrations fell below a predictable, threshold level. Collectively, these data indicate that TNT003 and TNT009 are potent and specific inhibitors of CCP activity and C3 fragment deposition in vitro and in vivo. These findings support the preclinical development of TNT009 for the treatment of CCP-mediated diseases including CAD. Disclosures: Panicker: True North Therapeutics, Inc.: Employment, Equity Ownership. Shi:True North Therapeutics, Inc.: Employment, Equity Ownership. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Tom:True North Therapeutics, Inc.: Employment, Equity Ownership. Strober:True North Therapeutics, Inc.: Employment. Sloan:True North Therapeutics, Inc.: Consultancy. Parry:True North Therapeutics, Inc.: Employment, Equity Ownership. Stagliano:True North Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Utility of A Single Widal Test in The Diagnosis of Typhoid Fever

Background & objectives: The clinical diagnosis of typhoid fever is difficult, as the presenting symptoms are often diverse and similar to those observed with other febrile illnesses. The definitive diagnosis of typhoid fever requires the isolation of Salmonella typhi or paratyphi from the patient concerned. Since patients often receive antibiotics prior to a confirmatory diagnosis, there is uncertainty that bacteria can be isolated from the blood cultures. Besides this, the facilities for blood culture are not always available or feasible. All these limitations have made Widal test the most utilized diagnostic test for typhoid fever. Many studies have produced data which had cast serious doubts on the value of the Widal Test and thus reappraisal of the role of a single Widal test is needed.Methods & materials: This study was carried out to determine the changes in clinical pattern of enteric fever. A total of 153 children, aged 0 to 14 years, diagnosed as typhoid fever (either positive blood culture for Salmonella typhi or paratyphi) were induced in the study. Of them, 86 children were with a definitive diagnosis of typhoid or paratyphoid fever as indicated by the isolation of S. typhi or S. paratyphi from the blood and 17 had negative blood culture but were clinically suspected of having typhoid fever. The control group was comprised of 50 children with non-typhoidal fevers The Widal test was carried out using rapid slide agglutination method and its accuracy was assessed by comparing the findings with that obtained through blood culture.Result: The mean age of the patients was 5.2 ± 2.8 years and the youngest and oldest patients were 0.7 and 14 years respectively and male to female ratio was roughly 1:1. Nearly one-quarter (24.6%) of the patients had been suffering from the disease for >10 days and the mean duration of illness was 8.2 ± 3.3 days. Widal Test result showed that an ‘O’ agglutinin titer of cut-off value e”1:40 gave a sensitivity of 87.2%, a specificity of 47.1%, a positive predictive value (PPV) of 89.2% and a negative predictive value (NPV) of 42.1%. The sensitivity and NPV decreased with the increase in titer levels and were 56.9% and 31.5% at cut-off value of e” 1:320, while the specificity and PPV increased with the increase in titer levels from 47.1% and 89.2% respectively at a titer of e”1:40 to 100% at a titer of e” 1:320. The ‘titer behaved in the same way as did the ‘O’ agglutinin titer. Similarly when H’ agglutinin was used the sensitivity and NPV decreased from 65% and 31.7% at a titer of e”1:40 to only 25% and 20% respectively at a titer of > 1:320, while specificity and PPV increased from 76.4% and 81.1% at >1:40 to 94.1% and 95.6% respectively at e” 1:320. When either ‘O’ or ‘H’ antibody titer of e”1:160 was used, a good sensitivity (71%), specificity (70.6%) and PPV (92.4%) resulted, though NPV decreased to 32.4%.Conclusion: The Widal test can be of diagnostic value when blood cultures are not available nor practically feasible.DOI: http://dx.doi.org/10.3329/bjch.v35i2.10377 Bangladesh J Child Health 2011; Vol 35 (2): 53-58

Secondary cold agglutinin disease associated with Hashimoto disease

Our case involves a 53 year old woman. Three years ago, she was investigated because of normal hemoglobin levels despite very a low erythrocyte count, which was revealed during the preoperative evaluation for ovarian cyst operation. The Direct Coombs test was found to be positive against complement and negative against IgG. Cold agglutinin titer was 1/448 (+). Due to the polyclonal IgM increase, secondary cold agglutinin disease (CAD) was considered but no factor could be found that would lead to cold agglutinin disease. During the post-operative follow-up, cold agglutinin titers increased with fluctuations in the patient. Twenty-four months after transabdominal hysterectomy and bilateral salpingooopherectomy operation, diagnosis of Hashimoto disease was made upon detection of subclinical hypothyroidism. No case of Hashimoto disease associated with CAD caused by polyclonal IgM has been reported until the present time.

Value of a Single-Tube Widal Test in Diagnosis of Typhoid Fever in Vietnam

The diagnostic value of an acute-phase single-tube Widal test for suspected typhoid fever was evaluated with 2,000 Vietnamese patients admitted to an infectious disease referral hospital between 1993 and 1998. Test patients had suspected typhoid fever and a blood culture positive for Salmonella typhi (n= 1,400) orSalmonella paratyphi A (n = 45). Control patients had a febrile illness for which another cause was confirmed (malaria [n = 103], dengue [n = 76], or bacteremia due to another microorganism [n = 156] or tetanus (n = 265). An O-agglutinin titer of ≥100 was found in 18% of the febrile controls and 7% of the tetanus patients. Corresponding values for H agglutinins were 8 and 1%, respectively. The O-agglutinin titer was ≥100 in 83% of the blood culture-positive typhoid fever cases, and the H-agglutinin titer was ≥100 in 67%. The disease prevalence in investigated patients in this hospital was 30.8% (95% confidence interval, 26.8 to 35.1%); at this prevalence, an elevated level of H agglutinins gave better positive predictive values for typhoid fever than did O agglutinins. With a cutoff titer of ≥200 for O agglutinin or ≥100 for H agglutinin, the Widal test would diagnose correctly 74% of the blood culture-positive cases of typhoid fever. However, 14% of the positive results would be false-positive, and 10% of the negative results would be false-negative. The Widal test can be helpful in the laboratory diagnosis of typhoid fever in Vietnam if interpreted with care.