Membrane-bound immunoglobulins and complement components on young and old red cells

In order to characterize changes in membrane-bound immunoglobulins and complement components, red cells (RBCs) were separated into young and old populations by simple centrifugation. Old RBCs had reduced mean corpuscular hemoglobin volume, increased mean corpuscular hemoglobin concentration, and reduced sialic acid. Using radioactive anti- antiglobulin techniques, old RBCs were shown to have more IgG, IgM, IgA, and C3d on their surfaces than did young RBCs; there was no increase on old RBCs of C3b, factor B, C4b, or C5. Similar results were observed with RBCs strongly coated with C3d in vivo from a patient with cold agglutinin disease. RBCs taken into ethylenediamine tetraacetate, washed thoroughly in saline, and then stored for prolonged periods in Alsever's solution or kept in autologous ethylenediamine tetraacetate plasma, at 4 degrees C, showed no increase in RBC-bound C3d with increased storage time. If, however, blood was taken into citrate- phosphate-dextrose and maintained at 4 degrees C in autologous plasma, a significant increase in RBC-bound C3d was observed in the mixed-cell population with prolonged storage time. Order donor blood units, taken into citrate-phosphate-dextrose and stored at 4 degrees C as packed red cells, showed higher levels of RBC-bound C3d in the mixed-cell population than did units stored for a shorter time. In no case did donor unit RBCs give a positive direct antiglobulin test on serologic testing with anti-C3d. The findings complement data already collected on membrane and cytoplasmic changes in aging RBCs and may contribute to an understanding of RBC senescence.     

Refrigerated storage of lyophilized and rehydrated, lyophilized human red cells

Human red cells (RBCs) were collected in CPDA-1 and then freeze-dried in lyoprotective solution. The lyophilized RBCs were then stored at -20 degrees C for 7 days. At the end of the storage period, the lyophilized RBCs were rehydrated and washed in dextrose saline. The washed, reconstituted, lyophilized RBCs were resuspended in final wash solutions of ADSOL, CPDA-1, or a special additive solution containing glucose, citrate, phosphate, adenine, and mannitol, and then they were stored at 4 degrees C for an additional 7 days. The main purpose of this study was to determine whether human RBCs can be lyophilized in such a manner that normal metabolic, rheologic, and cellular properties are maintained during rehydration and subsequent storage in standard blood bank preservative solutions. Our results show that reconstituted, lyophilized RBCs maintained levels of ATP, 2,3 DPG, lactate, and cellular properties that are equal to or better than those in control nonlyophilized RBCs stored for a comparable period in CPDA-1. Reconstituted, lyophilized RBCs stored at 4 degrees C after rehydration also show better maintenance of ATP, 2,3 DPG, and lactate than do control RBCs stored in the same preservative solutions for comparable periods.     

Characterization of various anti-Pr cold agglutinins

Forty -one samples of anti-Pr cold agglutinins (CA) were studied. The titers ranged from 4 to 32,000. As is the case with cold agglutinins of other specificities, immunoglobulin class M and k-type light chains predominated with anti-Pr CA. On the other hand, the few IgM lambda, IgG, and IgA found were associated preferentially with anti-Pr specificity. All anti-Pr CA were inhibited by human red cell membrane sialoglycoproteins. On the basis of an increased inhibition by sialoglycoproteins after periodate oxidation and carbodiimide treatment, respectively, three anti- Pr2 and five anti-Pr3 were found. Among 24 anti-Pr1 CA subclassified on the basis of agglutination with dog red cells, six were anti- Pr1h, 16 were anti- Pr1d , and two did not fit into the subclassification. Similar to the anti- Pr1h , - Pr1d subclassification, anti-Pr3 CA could be subclassified into anti- Pr3h , - Pr3d. Three anti-Pra examples were found. None of the anti-Pr CA was inhibited by N-acetylneuraminic acid; some (5/35) were inhibited by sialyllactose, NeuAc alpha 2–3 2–6 Gal beta 1–4Glc, in high doses (5.0- 20.0 mM).     

Waldenstrom's macroglobulinemia with an IgM paraprotein that is both a cold agglutinin and a cryoglobulin and has a suppressive effect on progenitor cell growth

BACKGROUND: A patient with Waldenstrom's macroglobulinemia was admitted to the hospital with fever, leg pain, and dyspnea. The patient had gas gangrene of the left leg that required above-the-knee amputation. Plasmapheresis was instituted to treat hyperviscosity. STUDY DESIGN AND METHODS: The patient's serum contained an IgM-kappa paraprotein, a cryoglobulin, and a cold agglutinin. The serum was studied. RESULTS: The patient's red cells typed as A1, Rh-positive. The direct antiglobulin test was negative. The serum contained a cold agglutinin with anti-Pr cold agglutinin specificity (titer 4096). Maximal thermal range was 30 degrees C. Following dithiothreitol treatment, the cold agglutinin activity disappeared. The serum IgM concentration in the tested sample was 62.3 g per L. The cold agglutinin titer in the supernatant after removal of the cryoglobulin was 256, and the IgM level was 0.31 g per L. Redissolving the cryoglobulin in a equivalent volume of saline resulted in a cold agglutinin titer of 4096 and an IgM level of 68.4 g per L. These results indicate that the cryoglobulin and the cold agglutinin are the same paraprotein. Serum protein electrophoresis using agarose gel and immunofixation of the serum revealed an IgM-kappa monoclonal band. Progenitor cell assays were performed by adding the patient's serum at final concentrations of 0, 1, 5 and 10 percent (vol/vol) to patient's and normal donor's peripheral blood mononuclear cells. Inhibition of burst-forming units- erythroid and colony-forming units-granulocyte/macrophage by the patient's serum was demonstrated. Appropriate controls and the use of the serum of another patient with Waldenstrom's macroglobulinemia did not suppress progenitor cell growth. The patient's serum inhibited colony formation in a dose-response fashion. CONCLUSION: Reports of cryoprecipitable cold agglutinins are rare. This case is unusual because the IgM-kappa paraprotein was also a cold agglutinin with anti- Pr specificity and erythroid and granulocyte-macrophage progenitor cell- suppressive properties.     

Fatal outcome in a patient with autoimmune hemolytic anemia associated with an IgM bithermic anti-ITP

BACKGROUND: Several cold autoantibodies (usually IgG) with IT specificity have been reported previously, as have autoantibodies with joint I and P blood group specificities (IP1, ITP1, iP1, IP). A fatal outcome associated with an IgM cold autoantibody of ITP specificity is reported. CASE REPORT: A 54-year-old man suffered from progressively severe cold autoimmune hemolytic anemia for 9 months. Hemoglobin concentration ranged from 6 to 7 g per dL (60-70 g/L) and reticulocytes from 3 to 5 percent (0.030-0.050). The direct antiglobulin test was weakly positive for IgM and strongly positive for C3d. The serum contained a cold agglutinin that reacted strongest with cord i red cells (RBCs) > adult I RBCs > adult i RBCs, which is consistent with IT specificity. The Donath-Landsteiner test was positive; the reaction was neutralized by globoside. The serum reacted weakly or was negative with RBCs from five group p blood donors, which suggests anti-ITP specificity. Dithiothreitol treatment of the serum abolished the cold agglutinin reactivity, which suggests that the anti-IT was IgM. The patient received > 40 RBC transfusions and failed to respond to oral steroids, oral cytoxan, high-dose pulse intravenous steroids, and plasma exchange at room temperature and at 35 degrees C. He died of sepsis following an unsuccessful trial of chlorambucil. Autopsy revealed unsuspected disseminated non-Hodgkin's lymphoma. CONCLUSION: Serologic studies are consistent with our patient's having a single IgM cold autoantibody with IT and P specificities (anti-ITP) and requiring both specificities on the same RBC to permit maximal antibody expression.     

Cold reacting antibodies recognizing antigens dependent on N− acetylneuraminic acid

Serum from a 75-year-old man with lymphoproliferative disease was found to contain a persistent cold agglutinin meeting most of the characteristics of examples of anti-Gd reported previously. It differed, however, in that it contained IgG and IgM components and its reactivity was not inhibited by sialyllactose. A search of random serums containing cold agglutinins was undertaken to determine the incidence of antibodies to antigens dependent on N-acetylneuraminic acid. Two further examples were found in 47 serums containing cold agglutinins. One of these also demonstrated a Gd or "Gd-like" specificity. The other appeared to be an anti-Pr.     

An unusual inhibition of an anti-P1

Tests for unexpected antibodies using commercially prepared red blood cells demonstrated no agglutination even though crossmatching and testing with saline washed commercially prepared reagent red blood cells identified an anti-P1. This antibody was inhibited when tested with red blood cells suspended in a modified Alsever's solution and several commercially prepared reagent red blood cell diluents. The inhibitory substance was identified as inosine. Hypoxanthine, adenine, and thymine also inhibited the anti-P1. Partial inhibition was produced by uracil, 5-methylcytosine, and xanthine. No other example of anti-P1 has been found to exhibit this unusual inhibiting characteristic.     

A potent cold autoagglutinin that recognizes type 2H determinant on red cells

An example of an IgM (lambda) cold autoagglutinin against a Type 2H determinant of the ABO blood group system is described. The direct antiglobulin test was positive due to C3d on the patient's red cells. The antibody in the patient's serum was active at 37 degrees C, and the degree of agglutination was almost the same at 37 and 4 degrees C. It agglutinated group O red cells preferentially, but not Bombay (Oh) red cells. The specificity was determined by adsorption with synthetic oligosaccharide immunoadsorbent.     

Transfusion support with RBCs from an Mk homozygote in a case of autoimmune hemolytic anemia following diphtheria-pertussis-tetanus vaccination

BACKGROUND: Autoimmune hemolytic anemia (AIHA) in children, although unusual, is often associated with recent infection. Several reports have identified the diphtheria-pertussis-tetanus (DPT) vaccination as a possible trigger for AIHA. STUDY DESIGN AND METHODS: Life-threatening AIHA was diagnosed in a 6-week-old infant 5 days after receiving a DPT vaccination. The patient required daily transfusion and/or exchange transfusion for 3 weeks. RBCs from an Mk homozygote were found compatible with the patient's autoantibody. Transfusion of RBCs from an Mk homozygote and later RBCs from an individual (K.T.) with a variant glycophorin, Mi.VII, were required to sustain the patient's Hb level until autoantibody production ceased, as evidenced by a fall in antibody titer and the patient's Hct returning to normal. RESULTS: The DAT was positive (3+) with only anti-C3 on presentation. An IgM cold reactive autoantibody with probable anti-Pr specificity and high thermal amplitude (37 degrees C) was identified in the serum. The DAT was no longer positive after transfusion with compatible blood. CONCLUSION: This case represents life-threatening AIHA in an infant, temporally related to a DPT injection and responsive to a combination of immunosuppression and transfusion of rare compatible blood.     

The role of carboxylic acids in EDTA-dependent panagglutination

The investigation of a patient blood sample showing a discrepancy between cell grouping and serum confirmation demonstrated a serum agglutinin which reacted with all red blood cells tested when exposed to EDTA. This reaction was 4+ macroscopic at room temperature, 2+ macroscopic with hemolysis at 37 degrees C in albumin, and 1+ macroscopic in the anti-human globulin phase. Agglutination was abolished following dithiothreitol treatment of the patient's serum or following saline washing of the EDTA-exposed test cells. The agglutination reaction was not limited to EDTA, but could be produced with polycarboxylic acids (citrate, L-tartrate, succinate) and monocarboxylic acids (acetate, lactate, propionate, valerate, butyrate). Non-carboxylic acids and low molecular weight ketones or alcohols failed in the agglutination reaction. This study reports an additional example of an IgM "EDTA dependent agglutinin" and demonstrates the dependence on carboxyl groups for its agglutinating activity.     

Resolution of serologic problems due to cold agglutinin mediated autoimmune hemolytic anemia and its transfusion decision

BackgroundAutoimmune hemolytic anemia (AIHA) is a rare disease characterized by hemolysis caused by autoantibodies against erythrocyte surface antigen. These antibodies can be classified as warm, cold, or mixed types.MethodsWe report two cases of cold agglutinin disease (CAD), which were eventually diagnosed owing to blood group discrepancy. Resolution was achieved after washing the red blood cells (RBCs) with warm saline and absorbing the autoantibodies at 4°C with the washed RBCs. We also assessed the patient''s condition and discussed the strategy of blood transfusion.ResultsThe first case occurred after postoperative chemotherapy for rectal cancer, and the other manifested with anemia from the outset. Direct antiglobulin tests were positive and revealed autoantibodies against C3d only. Cold agglutinin titration was performed, and the titers of both were 1:1024. Eventually, the patient''s condition stabilized without blood transfusion.ConclusionThe serological discrepancies observed in the blood transfusion department can successfully guide blood transfusion decisions in cases of CAD.     

Relationship of variable region genes expressed by a human B cell lymphoma secreting pathologic anti-Pr2 erythrocyte autoantibodies

To study the biology of cold agglutinin disease we previously established EBV-transformed B cell clones isolated from a patient with splenic lymphoma of an early plasmacytic cell type and immune hemolysis due to an anti-Pr2 cold agglutinin. These clones had an aberrant chromosomal marker identical to the patient's B cell lymphoma and each secreted IgMk anti-Pr2 similar to the pathologic autoantibody in the serum of the patient. In this study, we have further investigated the Pr2-specific autoimmune response through nucleotide sequencing of VH and VL region genes. We have shown that the seven clones share the same VDJ/VJ gene segments and junctional elements confirming their clonal origin. The VH sequences were 88% homologous to a VHI germline gene while the VL sequences were 97% homologous to a VkIII germline gene. Only 4 somatic mutations (3 silent and 1 conservative) were found in greater than 5,000 bp sequenced, suggesting that a low mutation rate existed. Based on a tumor mass of 10(12) cells and a minimum of 40 divisions, we estimated the somatic mutation rate to be 4.45 x 10(-5) m/bp/d. This somatic mutation rate is similar to those estimated for acute lymphocytic leukemia (pre-B cell) and chronic lymphocytic leukemia (intermediate B cell), but significantly lower than the mutation frequency in follicular lymphomas (activated B cell). We propose that the difference in somatic mutation frequency of a B cell tumor may be related to the stage of B cell differentiation. In addition, the low mutation frequency observed in the Pr2-specific B cell tumor may also reflect, in part, selection by autoantigen to conserve sIg structure and specificity.     

Anti-idiotypic antibodies specific for a pathologic anti-Pr2 cold agglutinin

The heterogeneity of human red cell (RBC) autoantibodies may be assessed by using anti-idiotypic antibodies. In this study, mouse monoclonal anti-idiotypic antibodies were produced against a pathologic RBC autoantibody with anti-Pr2 specificity. Epstein-Barr virus-transformed B-cell clones were established from a patient who had splenic lymphoma and associated immune hemolysis due to an anti-Pr2 cold autoantibody. Two of the eight clones producing this autoantibody were used to immunize mice for the establishment of hybridomas, and four monoclonal anti-idiotypic antibodies were isolated (2 IgG1 kappa and 2 IgM kappa). By the use of these anti-idiotypic antibodies, strong crossreactivity was seen on enzyme-linked immunosorbent assay with other anti-Pr2-producing clones from the same patient, but no cross-reactivity was seen with RBC autoantibodies from other individuals having anti-Pr or different specificities. Each of the anti-idiotypic antibodies inhibited hemagglutination (HA) by the patient's anti-Pr2 but failed to inhibit HA by antisera of a different RBC specificity. Cross-competition experiments indicated that all of the anti-idiotypic antibodies may recognize the same or a closely related idiotope on the anti-Pr2 autoantibody. These studies suggested that the four anti-idiotypic antibodies are directed against the same (or closely related) idiotypic determinant(s), unique to this patient's anti-Pr2 and located at or near the antigen-binding site. These anti-idiotypic antibodies may be useful tools for the study of this autoimmune response or for the development of immune therapeutic agents.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

IgA cold agglutinins recognize Pr and Sa antigens expressed on glycophorins

Three cases of IgA kappa cold agglutinins (CAs) were studied. One had anti-Pr1 specificity, one had anti-Pra, and one had anti-Sa. The CAs recognize O-glycans of glycophorins. The findings supplement previous data on anti-Pr1 specificities of four IgA kappa CAs. Because all IgA kappa CAs described recognize O-glycans of glycophorins, a close association between the CA IgA isotype and specificities for O-glycans becomes apparent. It is unlikely, however, that the striking association reflects interrelations between IgA CA structure and specificity, because anti-Sa specificity and all anti-Pr subspecificities were originally defined with IgM CAs.     

In vitro and in vivo measurements of gamma-radiated, frozen, glycerolized RBCs

BACKGROUND: Transfusion-associated GVHD results from the presence of viable lymphocytes in transfused allogeneic blood components. Viable immunocompetent lymphocytes have been detected in RBCs that were frozen with glycerol and washed before transfusion. STUDY DESIGN AND METHODS: The study reported here assessed the effect of irradiation on human RBCs frozen with 40-percent (wt/vol) glycerol and stored at -80 degrees C. In vitro and in vivo testing was done on human RBCs that were frozen with 40-percent (wt/vol) glycerol at -80 degrees C, with some units exposed to 2500 cGy of gamma radiation and others not irradiated, and that, after thawing and washing, were stored in a sodium chloride-glucose solution at 4 degrees C for 3 days before autologous transfusion. RESULTS: The glycerol-frozen RBCs treated with 2500 cGy before deglycerolization had a mean freeze-thaw-wash recovery of 87 percent and a mean 24-hour posttransfusion survival of 86 percent after storage for 3 days at 4 degrees C in a 0.9-percent NaCl and 0.2-percent glucose solution. For the nonirradiated units, the mean freeze-thaw-wash recovery was 85 percent and the mean 24-hour posttransfusion survival was 83 percent. CONCLUSION: These data show similar, acceptable results for RBCs frozen with 40-percent (wt/vol) glycerol at -80 degrees C and treated in the frozen state with 2500 cGy of gamma radiation and for RBCs that were not irradiated, all of which were washed and then stored in a sodium chloride-glucose solution for 3 days before autologous transfusion.     

Life-threatening, antiglobulin test-negative, acute autoimmune hemolytic anemia due to a non-complement-activating IgG1 kappa cold antibody with Pra specificity

A 21-year-old man with fulminant cold autoantibody hemolytic anemia (CAHA) was hospitalized with hemoglobinemia, hemoglobinuria, hemoglobin concentration of 3.3 gm per dL, a negative direct antiglobulin test (DAT) with polyspecific and anti-C3d reagents, a negative Donath-Landsteiner test, and a cold agglutinin titer of 80. He failed to respond to corticosteroids, multiple plasma exchanges, and cyclophosphamide; he required 54 transfusions in 10 days to maintain a hemoglobin concentration of 6.0 to 10.0 g per dL. He improved dramatically after a splenectomy was performed. The wide-thermal-amplitude cold agglutinin proved to be an IgG1 kappa antibody with Pra specificity. The patient's serum exhibited normal complement activation. When the DAT was carried out at 0 to 4 degrees C, the result was strongly positive for IgG; the indirect antiglobulin test at 0 to 4 degrees C was positive with the patient's serum diluted 1 in 640. Within 6 months, he was in complete remission and receiving no therapy. As compared with eight patients with CAHA that was exclusively IgG-mediated, this patient is remarkable for his requirement for many transfusions and for DATs that were consistently negative for C3d.     

A dual antiglobulin test for the detection of weak or nonagglutinating immunoglobulin M warm autoantibodies

BACKGROUND: Immunoglobulin (Ig)M warm autoantibodies (AABs) usually cause severe autoimmune hemolytic anemia (AIHA) and, in some cases, red blood cell (RBC)‐bound IgM cannot be detected. We describe a simple dual antiglobulin test (DDAT) for diagnosing such cases. STUDY DESIGN AND METHODS: A patient with erroneously suspected cold agglutinin syndrome was investigated. The direct antiglobulin test (DAT) was performed using standard techniques and dual (two stages) antiglobulin reagents (IgG rabbit anti‐human IgM, IgG goat anti‐rabbit IgG). RESULTS: A cold agglutinin syndrome was diagnosed initially, as the patient's serum was reactive with RBCs at a temperature of 28°C or less, and the DAT was strongly positive with anti‐C₃d. Six months later, the patient was reexamined at this hospital due to progressive hemolysis. His RBCs were found to be coated with IgM warm AABs that only became detectable using a DDAT, and his serum contained only a weak cold agglutinin. The hemolysis remained refractory to treatment with prednisolone and also prednisolone plus azathioprine, but gradually improved after treatment with prednisolone plus cyclophosphamide. CONCLUSION: Weak or nonagglutinating RBC‐bound IgM warm antibodies can be identified by the presented DDAT.     

Cryoprecipitation of an anti-Pr2 monoclonal IgM cold agglutinin in the presence of GM3 ganglioside.

The mechanism of cryoprecipitation of a monoclonal IgM kappa cryoglobulin (Mou) with a cold agglutinin activity of Pr2 specificity has been studied. By immunodiffusion this cryoglobulin reacted (by its Fab' fragment) with micellar GM3, a ganglioside bearing the Pr2 antigenic determinant. In contrast to previous reports that indicated a possible temperature dependent self-association of IgM molecules via an immunological interaction leading to cold precipitation, we could not detect any affinity of this cryoglobulin for IgM when we used passive hemagglutination or an indirect enzyme-linked immunosorbent assay (ELISA). However, a GM3-like ganglioside could be extracted, by drastic methods, from the cryoglobulin studied at 22 degrees C, whereas no GM3 was extracted from two control cryoglobulins. Some minor gangliosides (representing less than 25% of total amount of bound gangliosides) were also extracted from Mou cryoglobulin and these gangliosides were shown to crossreact with GM3, as they specifically bind to Mou cryoglobulin by ELISA. After cryoprecipitation the serum still contained a monoclonal anti-Pr2 IgM kappa. A GM3-like ganglioside could be extracted from this purified IgM, and cryoprecipitability could be induced by the addition of a minute amount of micellar GM3. These results suggest that Mou cryoglobulin circulates as an immune complex and that cryoprecipitation may depend on unique IgM-GM3 (or IgM-GM3 cross-reacting gangliosides) complexes.