Cold Agglutinin Antibodies Research Articles

Congenital cardiac surgery in a patient with cold agglutinins

Cold agglutinin disease is a type of autoimmune haemolytic anaemia caused by autoantibodies that bind to red blood cell antigens at a cold temperature and causes extravascular haemolysis and sometimes thrombosis also. Due to exposure to a lower temperature during open-heart surgery, the activation of haemolysis may occur in the presence of cold agglutinin antibodies. We present the management of a cyanotic child who has undergone open-heart surgery after the detection of high cold agglutinin titres during routine pre-transfusion screening.

Multifocal thrombosis with peripheral gangrene in a young boy: Mycoplasma infection triggered cold agglutinin disease

Cold agglutinin disease (CAD) is extremely rare in children. We report an 8-year-old boy who presented with gangrene of right foot with hypertension and absent lower limb pulses. Blood peripheral smear evidence of autoagglutination and falsely elevated red blood cell indices were suggestive of CAD and on subsequent investigations he was found to have high titres of cold agglutinin antibodies. He also had evidence of pneumonia on chest X-ray and serology for mycoplasma was positive. Computed tomography angiography showed multifocal thrombotic occlusion in bilateral popliteal arteries. He was effectively managed using antimicrobials, warm clothing, aspirin, anticoagulation and corticosteroids. He remains clinically well on follow-up and had no recurrence. CAD presenting with peripheral gangrene is extremely unusual. A careful look at peripheral blood smear gives an initial diagnostic clue. CAD triggered by infection is often self-limiting and requires supportive care.

Cold Agglutinin Disease Shows Highly Recurrent Gains of Chromosome 3, 12 and 18

Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia, caused by a distinct type of B-cell lymphoproliferative disease of the bone marrow. Anemia is mediated by binding of monoclonal cold agglutinin antibodies to the erythrocyte surface I antigen at temperatures below central body temperature, followed by agglutination and complement activation. These antibodies are almost exclusively encoded by IGHV4-34. We recently found recurrent KMT2D and CARD11 gene mutations in CAD (Malecka, et al 2017). We analyzed 12 CAD samples from the CAD-5 study (NCT02689986) (Berentsen, et al 2017) using Affymetrix OncoScan CNV Plus Assay and exome sequencing data in order to detect chromosomal aberrations. All samples were sorted using fluorescence activated cell sorting prior to analysis. Complete or partial gain of chromosome 3 (+3 or +3q) was detected in all samples (Table 1). Additionally, most cases showed either gain of chromosome 12 or 18 (9/12) (Table 1). Additional small regions of recurrent gain or loss were also detected in other chromosomes. The recurrent changes in least 4 samples are: +1p36.31-p36.13, -8p21.3-p21.2, +9q34.2-q34.3, +11q13.1-q13.2, +17q25.3, +21q22.3, +22q13.31-q13.33. Gains and losses of large parts of chromosomes are clearly visible in both Affymetrix OncoScan CNV Plus Assay and exome sequencing data (Figure 1). In contrast smaller changes are often not visible in exome sequencing data and therefore could not be fully validated. Further validation will be required for smaller chromosomal changes. Gain of chromosome 3/3q was characteristic for all CAD cases analyzed. Gain of chromosome 3 in CAD has previously been reported in 9 of 26 patients (Gordon, et al 1990, Michaux, et al 1995). Gain of chromosome 3 is not a highly recurrent finding in most B-cell lymphomas with the exception of marginal zone lymphoma (MZL). Of interest, gain of chromosome 12 and 18, demonstrated in our study, is also a feature of MZL. These findings, together with our previous findings (Malecka, et al 2017), suggest that the CAD-associated lymphoproliferative disease, although exclusively present in the bone marrow, might be related to MZL. The CAD-5 study assessed the efficacy of bendamustine plus rituximab in CAD patients. We explored whether a correlation existed between presence or absence of trisomy 12 and 18 with response to therapy. Although the series is small, we found a trend towards poorer response in patients with trisomy 18 compared to patients with trisomy 12. Furthermore, both patients without trisomy 12 or 18 had the best responses (Table 1). Despite the limited number of cases the Pearson correlation was statistically significant (p=0.02). However, more samples need to be analyzed to confirm these results. A correlation between response to therapy and presence of trisomy 12 or 18 has previously been demonstrated for other B-cell lymphomas. Trisomy 12 is frequent in patients with small lymphocytic lymphoma (28-36%) and in chronic lymphocytic leukemia (10-25%) and in the latter +12 is associated with intermediate prognosis (Autore, et al 2018). Trisomy 3 and 18 are found to be correlated with advanced stage extranodal marginal zone lymphoma (Krugmann, et al 2005). There is evidence from previous studies that trisomy 18 may be associated with upregulation of BCL2, an anti-apoptotic gene located on chromosome 18. Whether BCL2 is upregulated in CAD with trisomy 18 and whether it might be the cause of therapy resistance, needs to be investigated. Such a study is of importance since BCL2 inhibitors might be considered to improve treatment responses. We conclude that gain of chromosome 3 is a highly recurrent finding in CAD-associated lymphoproliferative disease. Further gain of chromosome 12 and 18 might be predictors of therapy outcome. These genetic findings are similar to what has previously been demonstrated in nodal and extranodal MZL, suggesting that CAD-associated lymphoproliferative disease, a disease of the bone marrow, might be related to MZL. Disclosures Berentsen: Mundipharma: Research Funding; Apellis, Bioverativ (a Sanofi company), Momenta Pharmaceuticals, and True North Therapeutics: Consultancy; Alexion, Apellis, and Janssen-Cilag: Honoraria. Tjønnfjord:Mundipharma: Research Funding.

A Rare Case of Mycoplasma Pneumoniae Infection Causing Cold-Agglutinin Autoimmune Hemolytic Anemia

SESSION TITLE: Student/Resident Case Report Poster - Chest Infections II SESSION TYPE: Student/Resident Case Report Poster PRESENTED ON: Tuesday, October 25, 2016 at 01:30 PM - 02:30 PM INTRODUCTION: Mycoplasma pneumoniae typically causes upper respiratory infections and atypical pneumonia and hemolysis can be an uncommon extrapulmonary manifestation. We present an unusual case of Mycoplasma pneumoniae pneumonia causing cold-agglutinin autoimmune hemolysis in an immunocompetent patient. CASE PRESENTATION: A 63-year-old Hispanic male with hypertension presented with non-productive cough for two weeks, associated with shortness of breath and fatigue. He was tacchycardic, tacchypneic, saturating 92% on room air, with a temperature of 102.1° Farenheit. Pulmonary exam revealed scattered rhonchi in the right lower lung field and diminished breath sounds in the left base. Laboratory investigations showed leukocytosis. Computed tomography of the chest showed areas of nonspecific groundglass attenuation in the right upper lobe and inferior right middle lobe along with enlarged hilar and subcarinal lymph nodes. IgM titer for Mycoplasma pneumoniae was 5081 units/mL (Ref < 770). Despite treatment with Ceftriaxone and Azithromycin, his hypoxia worsened as he became jaundiced with notable scleral icterus. His hemoglobin level decreased from 13.3 g/dL to 6.6 g/dL. Haptoglobin level was consistently low along with elevations in LDH, total and direct bilirubin. A peripheral blood smear revealed red blood cell agglutination with no evidence of spherocytes. Direct antiglobulin test was 3+ positive for C3d and weakly positive for IgG. Cold-agglutinin antibody titer obtained towards the end of his stay was 1:64, confirming the diagnosis of cold agglutinin autoimmune hemolysis. He finished a 14-day course of Azithromycin, received warmed IV fluids along with strict cold avoidance, leading to an improvement of his symptoms and hemoglobin. Two weeks after discharge, his hemoglobin continued to increase to 12.4g/dL. DISCUSSION: In 50 to 75% of Mycoplasma pneumoniae infections, IgM cold agglutinins are formed against erythrocyte I antigen but only rarely does it lead to hemolysis. The majority of erythrocyte destruction is extravascular as complement coated erythrocytes are phagocytosed by reticuloendothelial cells, especially Kupfer cells in the liver. In this case of autoimmune hemolysis, the patient’s symptoms improved with supportive treatment, cold avoidance, and an extended course of Azithromycin. Severe symptomatic hemolysis can be treated with Rituximab, cytotoxic agents or plasmapheresis. CONCLUSIONS: Mycoplasma pneumoniae is a well recognized cause of pneumonia but its association with cold-agglutin autoimmune hemolysis is rare; very few case reports exist around the world. Early suspicion and detection in the appropriate clinical setting can aid physicians in preventing further erythrocyte destruction in these patients. Reference #1: Han X et al. Mycoplasma pneumonia associated with hemolytic anemia: case report. Chinese J of TB and Resp Diseases. 2011 Nov;34(11):832-6. DISCLOSURE: The following authors have nothing to disclose: Robert Chao, Kayur Shah No Product/Research Disclosure Information

93. CANOMAD: Clinical and neurophysiological findings in two cases

The acronym CANOMAD (Chronic Ataxic Neuropathy, Ophthalmoplegia, IgM paraproteinemia, cold Agglutinin and Disialosyl antibodies) refers a rare immune-mediated peripheral neuropathy, whose clinical spectrum has still to be fully delineated. Herein we report the clinical and neurophysiological features of two CANOMAD patients. A 52-year-old man suffering from distal sensory symptoms and ataxia had received a first diagnosis of chronic inflammatory prominent-sensory polyneuropathy 11 years ago. High-dose i.v. steroid treatment resulted in paradoxical severe worsening with ophthalmoplegia and ambulation loss. Based on immunological and clinical–neurophysiological revaluation a diagnosis of CANOMAD was made. Since then IVIg periodic infusions had allowed good clinical remission up to-day, although minor degree of motor impairment and ataxia relapsed during last years. This 62-year-old man had a 5-year history of mild lower limb ataxia. More recently, a fluctuating bilateral ptosis was seen. Serum was positive for anti-disialosyl IgM antibodies and cold agglutinins. NCS/EMG showed a mild chronic sensory-motor neuropathy. Cortical somatosensory evoked potentials by tibial nerve stimulation at the ankle were deranged, whereas responses at the popliteal fossa and sural NCS were largely preserved. This suggests a proximal alteration of peripheral sensory conduction. These data further confirm the heterogeneous presentation of CANOMAD.

Primary Cold Agglutinin Associated Lymphoproliferative Disease: A B-Cell Lymphoma Of The Bone Marrow Distinct From Lymphoplasmacytic Lymphoma

Primary chronic cold agglutinin disease (CAD) is a rare hemolytic disease caused by the production of cold agglutinin antibodies that bind to erythrocyte I/i blood group antigens at low temperatures. The binding is mediated by monoclonal IGHV4-34-encoded cold agglutinins. CAD is a disease of older individuals with a median age of 76 years and accounts for about 15 % of all cases of autoimmune hemolytic anemia. The estimated incidence of CAD is 1-4/106 worldwide. The aim of this study is to better characterize the underlying lymphoproliferative disorder in CAD patients by review of bone marrow histology, immunophenotypic and genetic analysis. The bone marrow trephine biopsies of 54 patients with CAD showed circumscribed intraparenchymatous nodules with small monotonous monoclonal B-cells (median infiltration: 10% of marrow cells) with a CD20+, IgMs+, IgDs+, CD27+, CD5-/+, CD11c-/+, CD23-, CD38- immunophenotype. Neither plasmacytoid cytological features nor expression of plasma cell differentiation-associated transcription factors IRF4, BLIMP1 and XBP1 were expressed. However, a limited number of mature monoclonal IgM+, IgD- plasma cells were present outside the lymphoid nodules and were diffusely scattered throughout the marrow. Thus, the bone marrow in CAD shows a characteristic lymphoid infiltration pattern that is different from lymphoplasmacytic lymphoma that typically shows an admixture of lymphoid cells, plasmacytoid cells and plasmacells and that, not infrequently, shows a peritrabecular localisation. Of interest, the MYD88 L265P mutation, typical of lymphoplasmacytic lymphoma, was not detected (17/17 cases, sensitivity 3 %). Somatically mutated monoclonal IGHV4-34 gene rearrangement was demonstrated in eight patients with frozen samples (mean sequence homology 95.4%). Unexpectedly, no mutations of BCL6 intron 1 were demonstrated, except in one patient. BCL6 intron 1 mutations usually accompany immunoglobulin gene mutations in B lymphocytes that have matured in the germinal center. In conclusion, the histological, immunophenotypic and genetic properties of CAD are proof that CAD is a lymphoproliferative disease that is distinct from lymphoplasmacytic lymphoma with which it has previously been confounded. The disease likely originates from B lymphocytes that have not matured in the germinal center. Disclosures:No relevant conflicts of interest to declare.

Acute exacerbation of cold agglutinin disease during operation

Cold agglutinin, an autoantibody that combines with the red blood cell (RBC) carbohydrate antigen, is known to trigger hemolysis with agglutination at temperatures of 0-4℃ [1]. Cold agglutinin combined with RBC in a peripheral body exposed to a cold environment returns to the center of the body and combines with the complement; these processes form the mechanism for chronic cold agglutinin disease. However, these processes can result in acute aggravation due to stimuli such as infection and trauma [2]. The authors experienced hemolysis during an operation in a patient with cold agglutinin disease although his temperature was maintained above 35.6℃. A 67-year-old male patient diagnosed with bladder cancer was scheduled for a radical cystectomy with ileal conduit formation. He had been diagnosed with idiopathic cold agglutinin disease 20 years earlier. Hematologic conditions were stable without any medication. Hemoglobin was measured as 6.9 g/dl 2 days prior to the surgery. After 3 units of leukocyte-depleted RBC were transfused, the hemoglobin level increased to 9.9 g/dl. The cold agglutinin titer was 1 : 8192 and total bilirubin was 2.9 g/dl before the surgery. On the day of the surgery, the temperature of the operation room was raised to its maximum of 30℃. An esophageal stethoscope with a temperature probe was inserted after induction to read the core body temperature (BT), and a skin temperature probe was attached to the forehead to read the peripheral BT. The body of the patient, excluding the site of surgery, was covered in cotton blankets, and 2 air warmers were used. A normothermic (37℃) intravenous fluid administration set was used to elevate the temperature of all fluids. The inhalational gas supplied to the patient was set at 37℃ using a heated circuit. The core BT of the patient was 37℃ and his peripheral BT was 35.6℃ during the surgery. It was observed that the urine had a strong brown color [3], and at 60 minutes after anesthesia (30 minutes after the operation), blood tests showed hemoglobin at 11.0 g/dl, lactate dehydrogenase (LDH) at 494 IU/L, total bilirubin at 5.4 g/dl, and direct bilirubin at 2.1 g/dl. We decided that postoperative plasmapheresis was more appropriate than intraoperative plasmapheresis [4]. The estimated blood loss during the 600 minute surgery was 2800 ml, infused fluid HES 500 ml, and normal saline 4800 ml. Leukocyte-depleted RBC 6-units were transfused. Therapeutic plasmapheresis was executed for 180 minutes in the intensive care unit. The patient's blood was extracted through an arterial line and reinfused through a central venous line. Both lines were passed through fluid warmers. Therapeutic plasmapheresis was completed without any complications. The test results were as shown in Table 1. The hemoglobin level was stable without any significant decrease; therefore, the patient was transferred to the general ward on postoperative day 5 (POD#5). During the intensive care unit (ICU) stay, the temperature taken from the arm pit was above 38℃ for the first 2 days and remained at 37-38℃ until the fifth day. After transfer to the general ward, RBC 1 unit was transfused on each of the following days: POD#8, POD#10, POD#13, and POD#15. The temperature taken from the arm pit remained at 38℃. The patient was discharged on POD#16 with a hemoglobin level of 8.9 g/dl, and a month after the surgery (2 weeks after discharge), the hemoglobin measured at ambulatory care was 7.4 g/dl. There were no complications afterwards. Table 1 Changes in Laboratory Findings In this case, although the cold agglutinin antibody number was relatively high at 1 : 8148, the reactivity was not high due to the BT being at 36℃. The decrease in temperature during the surgery, however, might have caused the hemolytic reaction. It can be assumed that the low temperature (around 20-25℃) decreased the BT and consequently increased cold agglutinin reactivity. The high activity of cold agglutinin induced the hemolysis, the urine color change, and the high bilirubin level. Ulvestad et al. [5] reported that hemolysis occurred after infection and trauma in a patient with chronic cold agglutinin disease. The increases in pro-inflammatory cytokines IL-1α, IL-6, TNF-α, and IFN-γ in response to trauma can cause both an increase in BT and complement-mediated hemolysis, as well as possibly trigger an exacerbation of hemolysis. The patient in this case experienced hemolysis even when a relatively high BT (above 35℃) was maintained during the surgery. In summary, the maintenance of BT has been regarded as the most important step in the anesthetic management of cold agglutinin disease patients. However, because exposure to a relatively low temperature (25℃) could modify the cold agglutinin, hemolysis may occur even when the core BT is maintained at around 35.6℃. During surgery, the urine color must be carefully observed, and hemoglobin, direct bilirubin, and LDH must be measured to monitor for hemolysis.

Management of Cold Agglutinin–Immune Hemolytic Anemia in Pregnancy

Cold hemagglutinin disease is an acquired autoimmune hemolytic anemia caused by an immunoglobulin M autoantibody directed against the polysaccharide antigens on the red blood cell surface. This case presents the challenges surrounding the management of cold hemagglutinin disease in pregnancy. A pregnant woman in her thirties with type-2 diabetes, reporting shortness of breath and productive cough, was found to have anemia, reticulocytosis, bilirubinemia, positive direct Coombs test result, positive cold agglutinin antibody, and raised lactate dehydrogenase levels. As the infection screen and autoimmune serology results were negative, she was diagnosed as having idiopathic cold hemagglutinin disease. The management included keeping the patient warm and hydrated and treating the anemia with warm packed red blood cell transfusion. Cold hemagglutinin disease is a rare condition. Investigations to rule out infections help determine the diagnosis of cold hemagglutinin disease of unknown origin.

The cross-reactive idiotopes recognized by the monoclonal antibodies 9G4 and LC1 are located in framework region 1 of two non-overlapping subsets of human VH4 family encoded antibodies.

The monoclonal anti-idiotopic antibodies LC1 and 9G4 bind two non-overlapping sets of VH4 encoded antibodies. 9G4 exclusively binds VH4-21 encoded antibodies, while LC1 binds antibodies derived from VH4 family gene segments V71-2, V71-4, VH4-18, VH72-1 and V2-1. The VH4-21 gene segment is utilized by most cold agglutinin (CA) antibodies with I/i specificity, while antibodies encoded by other VH4 gene segments are associated not with CA disease, but primarily with rheumatoid-factor (RF) activity. We previously determined that the idiotope to which 9G4 binds in VH4-21-derived antibodies is located in framework region 1 (FR1). In the present study, by using mutational analysis involving individual framework- and complementarity-determining region exchanges between VH4-21- and V71-2-encoded antibodies, we have found that the idiotope to which LC1 binds in V71-2-derived antibodies also maps to FR1. The LC1 idiotope is heavy (H)-chain associated, but requires pairing with a light (L) chain for LC1 binding. Recombinant antibodies composed of a variety of kappa (kappa) and lambda (lambda) L chains paired with either a V71-2 or VH4-21 chain were produced in the baculovirus expression system. LC1 bound all of the kappa-containing antibodies but did not bind the V71-2-encoded H chain alone nor to the two lambda-containing antibodies. This experiment demonstrates that not all light chains exert equivalent influence on the conformation of the H-chain idiotope. These results indicate that the FR1 of VH4-encoded antibodies is immunogenic and suggest a physiological role of FR1 during an immune response.

Increased efficiency of binding of nascent C3b to the erythrocytes of chronic cold agglutinin disease.

The pathogenesis of chronic cold agglutinin disease (CCAD) has been enigmatic. To determine if abnormal erythrocyte membrane constituents might provide the stimulus for antibody production, we compared the electrophoretic pattern of radiolabeled membrane glycoproteins of four patients with CCAD to that of normal control erythrocytes. For the CCAD erythrocytes, fluorographs revealed the appearance of an abnormal band whose molecular weight was estimated at 126,000 D. Using two-dimensional gel analysis and immunoblotting techniques, it was determined that the 126,000 D glycoprotein consisted predominately of polymeric glycophorin-alpha. Previous investigations had suggested that abnormalities in glycophorin-alpha influence the functional activity of the complement system. When purified complement (C)3 was activated in the fluid-phase by cobra venom factor complexes, CCAD erythrocytes bound nascent C3b 7-27 times more efficiently than normal erythrocytes. Normal erythrocytes could be induced to manifest the appearance of the 126,000 D band, and the increased efficiency of binding of nascent C3b by incubation with CCAD serum or with the purified cold agglutinin antibody plus autologous serum, but not with the purified antibody alone or the purified antibody plus EDTA-chelated autologous serum. These studies demonstrate that the interactions of IgM cold-reacting antibody and complement with glycophorin induce changes in the biophysical properties of the erythrocyte membrane which modify subsequent interactions with complement.

The Effect of Hypothermic Cardiopulmonary Bypass on Patients with Low-Titer, Nonspecific Cold Agglutinins

The effect of hypothermic cardiopulmonary bypass (CPB) was studied in 5 patients with strongly positive cold agglutination at 4°C (experimental group) and in 10 controls. In the in vitro part of the study, the characteristics of the cold agglutinin antibodies in the experimental group included a low thermal amplitude (28°C or less), a low 4°C agglutination titer (1:32 or less), and non-specificity (non-anti-I and non-anti-i). The in vivo portion of the study revealed a fall in total urine and serum free hemoglobin in both groups on going on bypass, followed by a rise for the remainder of bypass. Statistical comparison between observed and expected total free hemoglobin for both groups showed a significant rise ( p < 0.05) after bypass, thereby indicating ongoing hemolysis. No statistically significant difference between preoperative and postoperative blood urea nitrogen and creatinine levels and gross neurological status was observed in the experimental group. It was concluded that the patient with nonspecific cold agglutinins whose antibody is characterized by a low titer, a low thermal amplitude, and a lack of clinical symptomatology can undergo hypothermic CPB without increased threat of a hemolytic or vascular occlusive crisis.

A Human Monoclonal Antibody Reacting with Merkel Cells: Immunofluorescence, Immunoperoxidase, and Immunoelectron Microscopy

A human monoclonal, mu, kappa, cold agglutinin antibody of the rare specificity Pr h (serum and proper eluates) was used in immunofluorescence and immunoperoxidase techniques and in immunoelectron microscopy on rabbit lip specimens. Pr h antibody strongly reacted with scattered cells in epidermis, which were demonstrated to be Merkel cells by electron microscopy; no nerve fibers were stained. In immunoelectron microscopy (IEM), a strong reaction was seen within the cytoplasm and around the granules. This is the first IEM staining of Merkel cells (MC) so far reported; it demonstrates the expression of a carbohydrate differentiation antigen in MC. The availability of a potent monoclonal antibody reacting with MC but not with neighboring epidermal cells in rabbit lip offers a new tool for the study of several aspects of MC biology, including antigenic properties and kinetics.

Cold agglutinin antibody in a hyperimmune erythrocyte antiserum.

AbstractTo examine whether an innocuous, nonbacterial-associated immunogen could generate a cold agglutinin autoreactive antibody response, rabbits were hyperimmunized with sheep erythrocytes. Of five animals immunized, one developed an autoreactive antibody population which agglutinated autologous and allogenic erythrocytes at temperatures below 37°. The cold agglutinin antibody activity was present in the IgM class immunoglobulin fraction since only the excluded volume of a Sephacryl S-200 serum fractionation, and purified IgM, contained detectable activity. The cold agglutinin antibody was hemolytic in the presence of guinea pig complement, since whole serum, and purified IgM, sensitized rabbit erythrocytes for lysis in a biphasic temperature hemolysis assay. In hemolytic inhibition assays, the cold agglutinin antibody was sugar specific. The relative sugar ligand specificity in this assay was shown to be N-acetylgalactosamine > melibiose > galactose > lactose. The hyperimmune anti-sheep erythrocyte se...

Properties of cold agglutinin and group carbohydrate-specific antibodies isolated from Group C streptococcal antisera

The relationship between erythrocyte autoreactive cold agglutinin (CA) antibodies and group carbohydrate-specific antibodies in hyperimmune rabbit Group C streptococcal antisera was investigated. The different antibody preparations examined were isolated from autologous erythrocyte, Group C carbohydrate, and α-GalNAc immunoabsorbents. Each population was subsequently tested for SRBC reactivity, RRBC autoreactivity, and carbohydrate and ligand reactivity, in hemagglutination assay, direct biphasic hemolytic and hemolytic inhibition assays, and radioimmunoassay. In absorption experiments, CA antibodies present in unfractionated serum and represented as purified IgG and IgM preparations, were reactive with Group C carbohydrate, but poorly reactive with α-GalNAc immunoabsorbent. In addition, CA antibody activity present in carbohydrate-eluted material, was absent in α-GalNAc-eluted material as determined by hemagglutination and direct hemolytic assay. By radioimmunoassay, carbohydrate-eluted and a-GalNAc-eluted streptococcal antibodies, and α-GalNAceluted BSA-α-GalNAc antibodies, exhibited similar reactivity with 125I-BSA-α-GalNAc antigen, and with GalNAc in radioimmunoassay inhibition experiments. In contrast to these results, both IgG and IgM CA antibodies exhibited a relatively low avidity toward 125I-BSA-α-GalNAc. Yet relative to the other antibody populations tested in radioimmunoassay inhibition experiments, CA antibodies did not exhibit a particularly significant difference in reactivity with GalNAc. However, recognition of other Gal-containing ligands, e.g. Mel and Lac, was restricted to the CA antibody preparations. These data suggest that CA antibodies present in Group C streptococcal antisera do not represent a higher affinity cross-reactive anticarbohydrate population, but instead perhaps represent cross-reactive carbohydratespecific antibodies produced in response to nonimmunodominant Group C carbohydrate determinants.

Reactivity with rabbit immunoglobulin G of cold agglutinins isolated from group C streptococcal antisera.

Cold agglutinin antibodies were isolated from group C streptococcal antisera by thermal elution from rabbit erythrocytes. These antibodies reacted with bovine submaxillary mucin, fetuin, immunoglobulin G, and the Fc fragment of immunoglobulin G in hemolytic inhibition assays. Further, in radioimmunoassay these antibodies reacted with the major glycopeptide fragment of rabbit immunoglobulin G. Affinity-purified group carbohydrate-specific antibodies reacted weakly with glycopeptide. These data suggest that certain populations of antibody in group C streptococcal antisera may participate in tissue reactivity via interaction with cell surface glycoproteins, including immunoglobulin G.

Factors affecting chain-interactions in immunoglobulins and their significance in cold-agglutinin activity

In competitive recombination experiments using non-alkylated heterologous H and L chains of monotypic immunoglobulins of different classes, subclasses and VL subgroups, an order of sequence in preferential reassociation of chains was proved. Preferential association on class and subclass level was found when H and L chains obtained from proteins of different classes and subclasses were used. The influence of the VL subgroup property was only found, when the chains were derived from proteins of identical subclasses. There was no significant difference between the attachment to H chains of autologous and heterologous L chains of identical subgroups. The influence of the exchange of L chains with heterologous L chains of different subgroups on the antibody activity of IgMK cold-agglutinins was also controlled. Molecules containing μ chains of IgMK cold-agglutinins and heterologous L chains agglutinated erythrocytes at +4°C. The fixation of isolated μ chains of IgMK cold-agglutinin antibodies to erythrocytes at +4°C was also demonstrated. The importance of the folded structure in preferential association of immunoglobulin polypeptide chains and significance of the overall conformation of the immunoglobulin molecules in antibody activity is discussed.

Studies of human sera with cytotoxic activity.

42 human sera showing in vitro cytotoxic activity of restricted or broad HL-A specificities with test human lymphocytes were studied for the molecular and immunoglobulin class of cytotoxic antibody using sucrose gradient separations, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration. Sera originated from patients with previous multiple pregnancies (19), multiply transfused patients (8), subacute bacterial endocarditis (4), systemic lupus (2), and human umbilical cord sera (9). In 32 of 42 instances, predominant cytotoxic activity was found in high molecular weight gradient fractions; however, DEAE chromatographic separations revealed cytotoxic activity in initial buffer fractions containing primarily gammaG globulin. Gradient separations of cytotoxic activity within initial gammaG DEAE fractions showed localization of cytotoxicity only in high molecular weight materials. Confirmation of high molecular weight gammaG cytotoxic activity was obtained by resistance to mercaptoethanol treatment, abolition of activity after absorption only with specific anti-gammaG antisera, and by the finding that high molecular weight cytotoxic activity in gradients or gel filtration run at neutral pH 7.4 became 7S when separations were rerun at an acidic pH of 4.0. Such 7S activity again became rapidly sedimenting when the same fractions were again rerun in gradients at neutral pH.19S gammaM cytotoxic activity was documented in a panel of 15 human sera containing anti-"I" cold agglutinins. In this instance the cytotoxic activity appeared to be related to the cold agglutinin antibody since it was mercaptoethanol sensitive and could be demonstrated in monoclonal antibody eluates containing primarily gammaM. No in vitro protection again cytotoxic effect was demonstrated when isolated human 19S anti-gamma-globulins were aded to the lymphocytotoxic assay system. With the exception of cold agglutinins, human cytotoxic antibodies appear to be primarily gammaG producing in vitro lymphocyte killing either as 7S gammaG globulin or as rapidly sedimenting aggregates or complexes of gammaG molecules.

Reaction of Cold Agglutinins with I Antigen Solubilized From Human Red Cells

Abstract The antigens reacting with cold-agglutinin antibodies and present in the red cell membranes of human red cells were found in the water-phase when the washed membranes were extracted with n-butanol. The presence of these antigens was demonstrated by agglutination inhibition, complement fixation, and antibody inhibition as determined by the C1 fixation and transfer test. Although antigen and antibody were not able to react at 37°C when the antigen was present in the intact cell, after solubilization, antigen and antibody reacted equally well at 37°C and at 0°C. The amount of anti-I inhibiting activity present in extracts from adult and cord cells was roughly the same, although the amount of antibody fixed to the intact cord cells was less with cord cells.

Cold-reacting Antibodies: Differences in the Reaction of Anti-I Antibodies with Adult and Cord Red Blood Cells

Abstract The reactions of anti-I cold-agglutinin antibodies with the red cells from normal adults (adult-I) and adult-i donors and from newborn infants have been investigated by the use of the C'1a Fixation and Transfer Test of Borsos and Rapp. Agglutination and high concentration of serum reduce the amounts of the C'1a fixation by these antibodies. Inhibition by serum is due to an inhibitor of C'1a. A single molecule of antibody is required for the fixation of one molecule of C'1a to either adult or cord cells. There was great variability in the amount of anti-I antibody and C'1a fixed by different adult cells and by different cord cells. Although the two ranges overlapped considerably, the amount of antibody fixed by cord cells was somewhat less than that fixed by adult cells. The difference between cord cells or "low-reactivity" adult-I cells and "high-reactivity" adult-I cells is not due to the presence of different or extra antigens on the latter since cord and "low-reactivity" adult I cells are able to absorb all antibody from the antiserum which reacts with "high-reactivity" adult-I cells. The difference between adult-I and cord cells (or adult-i cells) was reduced by pretreatment of the cells with papain and was increased by increasing the temperature at which the reactions were performed. These studies suggest that the difference between adult red cells and cord (or adult i) red cells with regard to the fixation of cold agglutinin antibody is due in large part to a difference in the "affinity" of the antibody and the antigen on the cell surface rather than to a major difference in the number of antigen sites.

Erythrocyte Acetylcholinesterase Deficiency in Paroxysmal Nocturnal Hemoglobinuria (PNH)—A Comparison of the Complement-Sensitive and Insensitive Populations

Abstract 1. The complement-sensitive and -insensitive cells of patients with paroxysmal nocturnal hemoglobinuria have been separated by differential lysis using a cold agglutinin antibody and limiting concentrations of complement. The erythrocyte acetylcholinesterase (AChE) activity of each population of cells was determined. 2. The membranes of the complement-sensitive cells characteristic of PNH completely lack AChE activity. 3. The membranes of the complement-insensitive cells usually contain less AChE than normal and the degree of decrease roughly parallels the degree of increased sensitivity to complement. 4. Lysis of normal cells by complement does not alter the AChE activity of their membranes. 5. The inhibition of AChE activity by neostigmine methylsulfate (Prostigmin®) does not increase the sensitivity of normal red cells to lysis by C’.