Hemolytic anemia associated with multiple autoantibodies and low serum complement

A 37 year old woman with extravascular hemolytic anemia had a positive Monospot test associated with positive antiglobulin and anticomplement Coombs' tests, cold agglutinins and warm autoantibodies. IgG-kappa (κ) antibodies, which reacted with all panel red cells at 37 °C, were eluted from her circulating red cells. However, neither immunoglobulins nor C3 was detected after her serum was adsorbed with heterologous red cell stroma at 37 °C and eluted at the same temperature in glycine buffer. In contrast, IgM-κ and IgM-Iambda (λ), IgG3-κ, IgG4-λ, IgA-λ and C3 were eluted at 37 °C from heterologous red cell stroma after adsorption with her serum at 0 °C. Thus, antibodies of several types, which were present in the patient's serum, reacted optimally with red cell antigens at low temperature. Cold-reactive IgG3-κ antibodies, which were also capable of interacting with red cells at 37 °C, probably accounted for the IgG-κ antibodies eluted from the patient's circulating red cells.The patient's serum C4 titers were decreased, with low normal to moderately depressed C3 and low normal C5, indicating that the anti-red cell IgM and/or IgG3-κ antibodies probably fixed complement.A localized cold stress test resulted in a transient increase in plasma hemoglobin and a decrease in serum C3 titer. These findings, and the beneficial clinical response obtained with small doses of prednisone, suggest that both the cold-reactive antibodies and the IgG-κ on circulating red cells were pathophysiologically significant.This is the first report of a patient with multiple red cell autoantibodies in whom serum complement component titers were determined in conjunction with characterization of the anti-red cell immunoglobulins. Subclinical infectious mononucleosis may have preceded the prolonged hemolytic episode. Clinical evidence of systemic lupus erythematosus has not appeared.     

Red cell alloimmunization in multitransfused patients and multiparous women

Purpose  To determine the incidence of alloimmunization against red cell antigens and the thermal amplitude and specificity of antibodies in multitransfused patients and multiparous women. Methods  Antibody screening was performed in 30 nontransfused orthopedic surgery cases (control), 504 multitransfused patients and 325 multiparous women. Antibody screening at 4°C, 22°C and 37°C was carried out in a saline phase, by indirect antiglobulin technique (IAT), using papain cystein, low ionic strength solution (LISS) and polyethylene glycol (PEG). Results  In multitransfused patients IgM antibodies were more frequently detected at 4°C and the IgG antibody incidence was 7.1% by enzyme method, 7.7% IAT, 9.4% LISS, 10.2% using PEG & 10.2% multiparous women. Bad obstetric history cases had significantly higher incidence of alloimmunization. The antibody specificity of antibodies was mainly in Lewis, Rh, Kidd and MN systems. Conclusion  Antibody screening before transfusion, at set time intervals after transfusion and during antenatal period is recommended.     

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

The antigens reacting with cold-agglutinin antibodies and present in the redcell membranes of human red cells werefound in the water-phase when thewashed membranes were extracted withn-butanol. The presence of these antigens was demonstrated by agglutinationinhibition, complement fixation, and antibody inhibition as determined by the C1fixation and transfer test. Althoughantigen and antibody were not able toreact at 37°C when the antigen waspresent in the intact cell, after solubilization, antigen and antibody reactedequally well at 37°C and at 0°C. Theamount of anti-I inhibiting activity present in extracts from adult and cord cellswas roughly the same, although theamount of antibody fixed to the intactcord cells was less with cord cells.

Submitted on March 25, 1970 Revised on June 18, 1970 Accepted on June 23, 1970     

Febrile temperatures control antineutrophil cytoplasmic autoantibody–induced neutrophil activation via inhibition of phosphatidylinositol 3‐kinase/Akt

Objective

Neutrophil activation by antineutrophil cytoplasmic autoantibodies (ANCAs) is central to the pathogenesis of the ANCA‐associated vasculitides. Febrile infections occur frequently during these diseases, often in the context of immunosuppressive treatment. Heat exposure may affect the underlying pathophysiologic processes of the vasculitis. In this study we tested the hypothesis that short‐term exposure to heat inhibits ANCA‐induced neutrophil activation.

Methods

After exposure to temperatures from 37°C to 42°C, human neutrophils were primed with either tumor necrosis factor α (TNFα) or granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and stimulated with monoclonal antibodies to myeloperoxidase or to proteinase 3. Respiratory burst activity was assayed using rhodamine and a nitroblue tetrazolium reduction assay. Specific inhibition experiments against p38 MAPK, ERK, and phosphatidylinositol 3‐kinase (PI 3‐kinase)/Akt, and Western blotting with phospho‐specific antibodies were used to identify key components in the antibody‐induced respiratory burst.

Results

A temperature‐dependent reduction in ANCA‐induced respiratory burst was observed over a range of heat exposures from 37°C to 42°C. Inhibition of human ANCA–induced neutrophil stimulation was significant at 40°C (after priming with 2 ng/ml TNFα, mean [± SEM] fluorescence intensity [MFI] 114 ± 12 at 37°C versus 53 ± 6 at 40°C; after priming with 20 ng/ml GM‐CSF, MFI 92 ± 16 at 37°C versus 35 ± 6 at 40°C; both P < 0.01). In the priming phase, the transient activation of the p38 MAPK, ERK, and PI 3‐kinase/Akt pathways by TNFα was blocked by prior exposure of the neutrophils to heat, but GM‐CSF–induced activation was unaltered by heat. However, in the second, antibody‐induced wave of kinase activation, exposure to heat inhibited only the PI 3‐kinase/Akt pathway, and these effects were independent of the priming agent used.

Conclusion

Short‐term spikes of modest heat abrogate ANCA‐induced activation of neutrophils via inhibition of PI 3‐kinase/Akt signaling. Febrile responses in ANCA‐mediated diseases may therefore have a physiologic purpose.

     

Autoimmune Hemolytic Anemia Associated with an IgG Cold Incomplete Antibody1

Abstract. Serologic studies are reported on a patient with severe autoimmune hemolytic anemia, whose red cells were strongly coated with IgG and with α2D component of C3. Direct and indirect Donath-Landsteiner reactions were negative. In addition to a typical IgM anti-I cold agglutinin of modest titer, the serum contained a lambda IgG incomplete antibody which bound more strongly to normal red cells at 4°C (1/64) than at 37°C (1/4). The IgG antibody did not require complement for binding but could bind complement and cause hemolysis with papainized red cells. Once bound, the IgG antibody did not dissociate appreciably at room temperature. The antibody eluted poorly from the patient's cells at 56°C but was readily dissociable from cell stroma at pH 3.5. The eluate exhibited no blood group specificity with a diagnostic red cell panel. In particular, equally strong reactions were obtained with O, A₂, B, A₁B, P + P₁-negative, Rh-null, adult and cord red cells. Reactivity was not impaired by prior papainization of the cells. Although the patient improved following splenectomy, neither IgG nor IgM antibodies were detectable in a concentrated splenic extract. The properties of the present cold IgG incomplete antibody are compared with those previously reported; possible clinical implications of these antibodies are briefly discussed.     

A Case of Cold Haemoglobinuria with Later Sarcoidosis Treatment with Plasmapheresis and Immunosuppressiva

A case of severe but transient haemolytic disease occurring after a febrile episode is described. The thermal amplitude of the haemolysin was high during the acute phase, since the autoantibody fixed complement at 31° C. After 4 months complement fixation could exclusively be demonstrated at 4° C. The patient was treated in a room heated to 30–32° C. The treatment consisted of prednisone and azathioprine and during the acute phase plasmapheresis was attempted in order to reduce the antibody concentration. However, the haemolysis decreased when the thermal amplitude of the antibody diminished. 1 year after termination of therapy, she developed sarcoidosis.     

Temperature dependence of the calcium paradox in the isolated working rat heart: Discrepancy between functional recovery and enzyme release

We studied the effects of brief (2 min) acalcemic perfusion at various temperatures in isolated working rat hearts. Recovery of aortic flow, coronary flow and lactate dehydrogenase (LDH) release were measured upon reperfusion at 37°C. With acalcemic perfusion at 37°C, there was complete absence of functional recovery and impaired coronary flow. Acalcemic perfusion at 15°C and below resulted in complete recovery of function and coronary flow. Acalcemic perfusion between 20 and 30°C resulted in partial recovery of function and complete recovery of coronary flow.In contrast to this relationship of functional recovery to temperature, LDH release occurred only at 37°C. Thus, functional recovery does not correlate well with enzyme release over a range of acalcemic perfusion temperatures.     

Quantitative evaluation of antibody-dependent lymphocyte-mediated lysis of human red cells

The ability of lymphocytes to lyse human red cells coated with anti-D antibody was assessed by measuring 51 Cr release from labeled red cells incubated with peripheral blood leukocyte suspensions from 12 normal donors. Mixed mononuclear cell suspensions (containing monocytes and lymphocytes) from all donors produced lysis of sensitized red cells. Treatment with carbonyl iron reduced monocyte concentration to less than 1.2% in all donors, as measured by morphologic criteria, esterase staining and ingestion of latex particles. Lysis of red cells following monocyte depletion was markedly reduced in 8 of the 12 donors. Despite depletion of monocytes, unchanged or increased lysis was noticed with the leukocytes of the remaining 4 donors. This lysis was due to lymphocytes, not to residual monocytes. If target red cells were treated with papain or trypsin prior to sensitization, marked lysis occurred with lymphocytes of all donors, including those which did not lyse unmodified red cells. Direct cytolysis of sensitized red cells during contact with small lymphocytes was recorded using microcinematography, which confirmed the role of lymphocytes in mediating lysis. Lymphocyte-mediated lysis of red cells increased with mounting levels of antibody sensitization regardless to prior treatment with papain. Papain increased antibody coating per red cell, yet lysis per molecule of antibody bound was also increased. Lysis was inhibited by IgG1 and IgG3 in the fluid phase but not by IgG2 or IgG4. At an equivalent level of antibody sensitization lysis was augmented by concurrent coating of the red cells with C3b, C3d and/or C4b, though these components could not produce lysis in the absence of antibody coating.     

Citrate-Dependent Auto-Antibody Causing Error in Blood Grouping

Background and objectives: Errors in blood grouping are occasionally due to anomalous antibodies or extraneous materials in the reagents. We investigated the case of a volunteer blood donor who was originally grouped as AB, Rh-positive, by slide grouping. However, her washed red cells showed group B, which agreed with her reverse serum grouping. Materials and methods: Standard serologic techniques were employed throughout. Results: Investigation revealed an unusual autoantibody in the donor's blood that preferentially agglutinated the red cells in the presence of citrate. Because the grouping reagents contained citrate, this led to a false grouping when whole blood was tested rather than washed red cells. The antibody reacted at 37 °C and at 4 °C, but failed to agglutinate red cells when 0.1 M solution of monosaccharides was added to the reaction mixture. The antibody agglutinated all red cells except 6 examples of the ‘Bombay’ phenotype included in the panel. The antibody was neutralized by H secretor saliva, and its activity was abolished by DTT reagent, indicating its possible IgM nature. Conclusion: These findings suggest that this anomalous antibody has anti-H specificity.     

Quantitative changes in myocardial nucleic acids and protein during normothermic and hypothermic pulsatile perfusion with bloodless fluid

Pulsatile perfusion of rabbit hearts with bloodless fluid was performed for 2 to 6 h at normothermic and hypothermic temperatures. All perfused hearts became edematous with the maximum gain in weight occurring at 37°C and the minimum at 5 to 15°C perfusion. Concomitantly, the dry weight fraction of hearts decreased, with the minimum decrease noted at 15°C and the maximum at 37°C perfusion temperature. The DNA content of perfused hearts declined, with the sharpest drop registered at 2-h perfusion and the maximum retention of DNA observed at 25°C. Protein content of myocardium in perfused hearts decreased with the minimum loss observed during 15°C perfusion. In contrast to DNA and protein, the RNA levels were essentially unchanged. It is concluded the pulsatile perfusion within a 15 to 25° temperature range insures maximum retention of myocardial dry weight fraction, DNA, protein and RNA.     

Studies on the Ultrastructure of Pseudopod Formation in Human Blood Platelets.

A method for quantitative evaluation of blood platelet pseudopod formation is presented. Pseudopod formation was studied in whole blood incubated for 0, 5, 10, 20 and 40 minutes at + 4°, + 15° or + 37° C. Samples were anticoagulated with EDTA or sodium citrate, or incubated without anticoagulant. In instantly fixed blood, platelets showed a pseudopod index of about 0.5. In samples incubated at + 4° C., the pseudopod index rose to about 3 after 20 minutes. After 40 minutes there was no further increase. At + 37° C. the pseudopod index did not increase during the first 20 minutes of incubation, but after 40 minutes it had risen to levels almost similar to those of the + 4° samples. EDTA and sodium citrate exerted no influence upon pseudopod formation at any temperature level tested. Mechanical forces operating during the preparation of platelet rich plasma caused pseudopod formation and enhanced temperature induced pseudopod formation. These results support the theory of Bull & Zucker (1965) that blood platelets maintain their lenticular shape by energy requiring processes.     

Complement-Independent Lysis of Human Red Blood Cells by Cold Hemagglutinins

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.     

A Stable Reagent System for Screening and Identifying Red Blood Cell Irregular Antibodies: Application to Commercial Antibodies

Objective: Development of a new solid-phase system for screening and identifying irregular red cell antibodies. Materials and Methods: Red blood cell membranes were prepared by a semi-automated procedure in which the hemolysate solution was passed through a hollow-fiber system. The membranes were fixed to the solid phase (microtiter plates) by centrifugation and incubated with 8% fat-free milk. Antibodies added to the microtiter plate were detected by antihuman antibodies adsorbed onto yellow latex particles. Results: The system had good sensitivity (titer <1); 97% of anti-D samples were detected. The detection system was stable for 6 months at 4°C. Conclusion: This stable-antigen solid-phase system readily detects and identifies red cell antibodies that are important in transfusion.     

Paroxysmal cold haemoglobinuria in an adult with chicken pox

Paroxysmal cold haemoglobinuria (PCH) is an autoimmune disorder characterized by intravascular haemolysis causing haemoglobinuria. It is due to a biphasic haemolysin known as the Donath-Landsteiner antibody, which binds specifically to the P antigen of red blood cells at low temperatures, leading to complement activation and red cell lysis at 37 degrees C. PCH is a rare disease which predominantly affects the paediatric population, occurring mostly during viral infections. We report on what is possibly the first case of PCH in an adult to be precipitated by chicken pox infection.     

Absence of B antibody in a blood group A person

Abstract. A healthy Caucasian woman who possessed blood groups A₂H(O) on her red cells as well as in her saliva and serum had no anti-blood group B ‘complete’ or ‘incomplete’ agglutinins nor hemolysins as determined either on whole serum or its concentrated β-γ-globulin fraction. No anti-B antibody was stimulated by immunization with highly active human blood group B glycoprotein. No blood group B antigenic determinants were demonstrable on the proposita's red cells or in her saliva by any of numerous serological and enzymatic methods at various red cell concentrations between 2 and 37 °C, nor by the highly sensitive immunoglobulin-latex test. Immunization with the proposita's red cells did not elicit anti-blood group B antibodies in chickens or rabbits. Blood groups A and O but not B occurred in members of this woman's family which was studied through 3 generations.     

REVERSE RELATIONSHIP BETWEEN MITOGEN ACTIVATED PROTEIN KINASE AND HUMAN PLATELET AGGREGATION

The role of MAPK in platelets was investigated. In human platelets maintained at 4°C for 2 hr, the MAPK activity increased (～ 2 fold) when compared to those maintained at 37°C. When aggregation was monitored under these conditions, the platelets maintained at 4°C or 15°C showed an 85% and 71% decrease respectively to thrombin (0.5 U/ml for 1 min) induced aggregation. When the platelet cytosol was maintained at 4°C and assayed for MAPK activity, the MAPK activity decreased significantly, indicating that the observed effects are seen in intact platelets only, and are not due to temperature effects on the assay. When platelets maintained at 4°C or 15°C (for 2 hrs) were transferred to 37°C, the MAPK activity decreased to levels observed in platelets maintained throughout at 37°C and was thus reversible. Therefore, it is concluded that a possible reverse relationship between MAPK and platelet aggregation plays a role in platelet responses.     

The stability of the I, HI, and H blood group antigens during storage at 4 degrees C

Abstract. In agglutination experiments using antisera of human origin, the I, HI and H erythrocyte antigens were shown to be quite stable on red cells that had been collected as clotted blood and in ACD-B solution and maintained at 4°C for periods as long as 28 days. These blood group antigens were also well preserved on red cells that had been frozen at -150°C in the vapor phase of liquid nitrogen and, after recovery from the frozen state, were maintained at 4°C in a dextrose-electrolyte solution for 7 days.     

Detection of Irregular Red Cell Antibodies: More than 3 Years of Experience with a Gel Technique and Pooled Screening Cells

Background and objectives: The purpose of this study was to evaluate more than 3 years of experience with a gel technique in combination with pooled screening cells for the detection of irregular red cell antibodies. Materials and methods: Conventional serologic methods were used for blood typing, antibody screening and cross-matching until the end of 1992. We introduced the gel technique as a routine assay for antibody detection and identification in 1993. Results: After the tube technique had been abandoned, the number of false-positive antibody screening tests was reduced by 71%, positive antibody screening tests by 33%, enzyme agglutination by 100% and rouleaux reactions and cold-reacting antibodies by more than 50%. There was a 40% increase in first-time detection of clinically relevant antibodies. We saw no increase in delayed haemolytic transfusion reactions. Conclusions: For the detection of irregular red cell antibodies, pooled screening cells in combination with a gel technique are at least as efficient and safe as a conventional tube technique with unpooled test cells.     

Acute autoimmune hemolytic anemia due to a low molecular weight IgM cold hemolysin associated with episodic lymphoid granulomatous vasculitis

The case of a patient with a 3 year long illness characterized by episodic localized lymphadenitis and concurrent acute autoimmune hemolytic anemia is reported. Histopathologically the lymphoid disease was granulomatous necrotizing vasculitis of the Wegener type. The acute hemolysis occurring with these episodes was associated with extreme erythrophagocytosis in the peripheral blood without hemoglobinuria. The unique autoantibody resembled the cold agglutinins of lymphoproliferative diseases in that it was an immunoglobulin M (IgM) with only kappa light chains and had anti-I specificity. However, the antibody had no agglutinating ability, demonstrated biphasic thermal requirements for in vitro hemolysis (cold, then warm, incubation) and was of approximately 7S size. Despite the requirement for incubation at less than 10 °C to produce in vitro hemolysis, clinical episodes of severe hemolytic anemia were unassociated with cold exposure. An excellent response to cortico-steroids contrasted with the typical experience with cold-reacting red cell autoantibodies.     

Antibody-Dependent Monocyte-Mediated Cytotoxicity.

Monocyte mediated lysis of human type B-erythrocytes coated with heat-inactivated anti-B serum was measured as ⁵¹Cr-release. Preincubation of monocytes with heat-inactivated normal AB serum inhibited cytolysis while a strong stimulation of cytolysis was seen with fresh AB serum. These effects of serum were reversible upon cell washing. Preincubation of monocytes with hyperimmune human serum against type B erythrocytes did not alter the cytolysis. After incubation at 37° C of monocytes with immune complexes formed of human albumin and rabbit anti-human albumin a dose-related inhibition of cytolysis was observed. This inhibition was equally expressed at different albumin to anti-albumin ratios. Pretreatment with immune complexes at 4°C had the same effect on cytolysis as pretreatment at 37° C. The effect of immune complexes on monocyte-mediated cytolysis was resistant to repeated cell washing. After incubation of pretreated monocytes at 37°C for up to 18 h some of the inhibition disappeared, but without normalization. Platelet depletion of monocyte suspensions resulted in a significant increase of cytolysis, and addition of washed platelets reinduced a decrease of cytolysis, which correlated with the number of platelets added.