Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies

The physiological breakdown of C3 has been studied using monoclonal anti-C3 antibodies, and it has been found that the later stages of this process--the breakdown of C3bi--is more complex than had previously been recognized. C3bi is the reaction product produced from C3b by the action of factor I which, in the presence of factor H, produces a double cleavage in the alpha chain of C3b. It is here reported that, both on cells and in the fluid phase, the breakdown of C3bi in serum gives rise to two products: C3c and the product previously described as alpha 2D, which we now propose to designate C3d,g. Alpha 2D differs from C3d in that it contains an additional fragment of approximately 8,000 mol wt that carries the antigenic determinant for the clone 9 monoclonal anti-C3 antibody. C3g cannot be precipitated by anti-C3 antisera and therefore behaves as a uni- or bideterminant antigen. The cleavage of C3d,g to C3d and C3g does not occur in sterile serum. It is also still uncertain what enzyme cleaves C3bi to C3c and C3d,g in plasma. Plasmin can do so in vitro, but plasminogen-depleted serum can still produce the cleavage. The antigenic determinant recognized by clone 9 in C3 is not exposed in C3 or C3b, but appears as a neoantigen in C3bi (and in C3d,g). Anti-C3g therefore is a potentially useful ligand for detecting complement-activation products. C3g represents a new, highly anionic C3 fragment and seems not to be identical with the C3e fragment described by others.     

Purification and use of the C3d subunit C3

A method is described for the preparation of C3d from fresh-frozen CPD plasma. A redissolved EDTA euglobulin precipitate formed from defibrinated plasma is chromatographed on DEAE cellulose. The C3-rich peak is further chromatographed by stepwise elution from hydroxy-apatite. The highly purified C3 and C3b so obtained are then treated with commercial C3b inactivator. G-200 sephadex filtration of this material produces a low molecular weight peak containing C3d greater than 95 per cent purity. The purified C3d, which contains negligible carbohydrate, has a molecular weight of 28,000 (based on SDS polyacrylamide gel electrophoresis). When added at a final concentration of less than 70 mug per ml, it completely inhibits the anti-C3d hemagglutinin reactivity of a potent anti-C3d antiglobulin serum but does not inhibit sera with reactivities against C3c, C4b, and C5b. Two of three rabbits hyperimmunized with purified C3d produced antisera exhibiting only anti-C3d precipitin activity. After absorption of heterologous antibodies, the antisera strongly agglutinated C3d-coated RBC, failed to agglutinate RBC glutinated C3d-coated RBC, failed to agglutinate RBC coated only with C4, and reacted weakly against strongly anti-Rh coated RBC. The latter reactivity was readily absorbed by whole IgG. Preliminary studies with 125I-labeled C3d indicate its potential value in assessing potency of anti-C3d reagents.     

The significance of red cell bound complement components in development of standards and quality assurance for the anti-complement components of antiglobulin sera

Human red blood cells, sensitized with complement in vivo and by a variety of methods in vitro, (e.g. blood group antibody, low ionic strength, alternate pathway), were tested with a battery of anti-complement sera (anti-C3, -C3c, -C3d, -C4, -C4c). Red blood cells could be prepared by relatively simple methods to yield cells sensitized with C3 and C4, C3 but not C4, C4 but no C3, C3d with no C3c and C4d with no C4c. These cells are suitable for standardization and quality assurance of antiglobulin sera (AGS). Anti-C3d is necessary for optimal detection of sensitization of red blood cells by complement in vivo by the direct anti-globulin test (DAT). Anti-C3d may also be optimal for the indirect antiglobulin test (IAT) especially if incubation periods greater than one hour are employed. Potent anti-C4 and anti-C3 antisera made in the authors' laboratory resulted in numerous weakly positive antiglobulin tests when testing red blood cells from refrigerated clots (especially anti-C4) but red blood cells from refrigerated anticoagulated segments gave negative results. When red blood cells were incubated in normal serum at room temperature (as in the room temperature phase of a compatibility test), some positive results were again obtained with the potent anti-C4 and anti-C3 antisera. However, one commercial antiglobulin serum containing anti-complement antibodies that were at least as potent as any other commercial antiglobulin serum gave uniformly negative results under the above conditions. Anti-C4 antibodies may be omitted from antiglobulin sera without decreasing the efficacy of such antisera to be used in compatibility testing. Thus, positive results in the compatibility test due to detection of clinically insignificant cold antibodies in the IAT by the anti-complement antibodies in AGS, may be avoided if anti-C4 is omitted or is in low concentration and if the concentration of anti-C3d is carefully standardized. A higher concentration of anti-C3d could be used for compatibility tests if red blood cells from anticoagulated segments were used instead of those from clots and if a separate tube were used for the IAT at 37 C rather than using one tube for both room temperature and 37 C incubations.     

Serologic comparison of a monoclonal anti-C3d with commercial antiglobulin reagents

The serologic reactivity of a monoclonal anti-C3d and commercial "monospecific" anti-C3 and polyspecific antiglobulin reagents was compared. The testing consisted of a direct antiglobulin test performed on donor and patient cells and on in vivo and in vitro complement- coated cells; an indirect antiglobulin test was performed with low- ionic-strength solution crossmatches. The monoclonal anti-C3d demonstrated reactivity very similar to that of the commercial heterogeneous rabbit anti-C3d; it gave relatively few weak "false positive" reactions with normal donor and patient red blood cells, and comparably strong reactions with in vitro and in vivo C3d-coated red blood cells. The results suggest a promising future for monoclonal antiglobulin reagents.     

Anti-C3d Antiglobulin Reagents. I. Characteristics of the Anti-C3c and Anti-C3d Responses During Hyperimmunization in Rabbits

Antiglobulin sera designed to detect erythrocyte coating by the third component of complement (C3) should contain primarily antibody to the C3d (α2D) antigenic grouping on the C3 molecule. During immunization, animals respond independently to the C3c (βlA) and C3d antigens. The present study reports quantitative serial precipitin and antiglobulin measurements of anti-C3c and anti-C3d responses in 12 rabbits during a four and one-half month hyperimmunization program and a subsequent six month follow-up period. Anti-C3c precipitin responses occurred earlier and were sustained longer than were anti-C3d precipitin responses. There was a general correlation of the strength of anti-C3d precipitin and antiglobulin responses among serum samples from a single rabbit, but not among sera from different rabbits. While most rabbits developed strong anti-C3c and anti-C3d precipitin and antiglobulin reactivities at the peak of their immune response, five of five commercially manufactured goat anti-C3 sera exhibited strong anti-C3c reactivity with little or no anti-C3d reactivity. The implications of these observations for production of anti-C3 antiglobulin reagents are discussed.     

Differences in specificities of anti-C3d sera raised to C3d antigens prepared in different ways

Anti-C3d sera were raised to different C3d antigens or to the same C3d antigen by different methods. Although identical by immunoprecipitation studies, the various anti-C3d sera showed differences in specificities against bound C3d antigen. Such differences were observed wih red blood cells coated with C3d in vivo and in vitro. Antisera made to the C3d− KAF antigen detected fewer molecules/cell on C3d−tryp cells than did antisera made to the C3d−tryp antigen. The converse was true for C3d− KAF cells. "Saturation" experiments indicated that different anti−C3d detected different "subpopulations" of bound C3d. C3d bound to red blood cells in vivo was, in at least one case, detectable by some anti- C3d sera but not by others. Such differences in anti-C3d specificity may be important in determining the optimal characteristics of anti-C3d antiglobulin serum for routine laboratory use.     

Radioimmunoassay evaluation of anti-C3d reactivity in broad spectrum commercial antiglobulin reagents

The study was undertaken to assess the potential of a labeled C3d radioimmunoassay method as a standardization criterion for antiglobulin reagents containing anti-C3d reactivity. Over an 18-month interval, four different lots of broad-spectrum antiglobulin reagents were purchased from each of seven United States manufacturers. Anti-C3d antibody concentration and Ko in each lot were assayed initially and at four-month intervals over 16 to 21 months storage at 4 degrees C. The results permitted 1) comparison of anti-C3d antibody concentrations among different manufacturers, 2) comparison among different lots from the same manufacturer, and 3) assessment of stability of anti-C3d during storage. In addition, serial dilutions of reagents in each manufacturer's diluent were compared for their anti-C3d agglutinating properties by the spin-antiglobulin method against red blood cells (RBC) coated by C3d in vitro and against in vivo C3d-coated RBC from 16 patients. Most reagents were shown also to contain anti-C3c, anti-C4c and anti-C4d reactivities when tested against suitably coated RBC. Anti- C3d antibody concentrations ranged from approximately 1 to 3.5 microgram/ml for 27 of the 28 reagents and were stable over 16 to 21 months of storage. Anti-C3d agglutination titer scores showed a general correlation with anti-C3d antibody concentrations when tested against in vitro C3d-coated RBC; correlations were less good against C3d-coated cells from patients. The radioimmunoassay provided reproducible objective measurements and appeared to have merit as a standardization criterion.     

Anti-C3d Antiglobulin Reagents. II. Preparation of an Antiglobulin Serum Monospecific for C3d

Three approaches are described to the preparation of a potent antiglobulin reagent monospecific for the C3d component of C3: 1) attempts to absorb out the anti-C3c component of a bisperific anti-C3c, C3d serum, 2) attempts to isolate by affinity chromatography purified C3d to be used for animal immunization, 3) animal immunization with a relatively crude filtration fraction of aged serum which contained C3d but lacked C3c. Problems encountered with the first two approaches are described and discussed. Preparation of a potent anti-C3d serum by the third approach is documented, as are methods employed to assess the specificity and potency of the reagent.     

Partial characterization of physiologically generated C3 components expressing C3d but not C3c epitopes

Techniques for the quantification of C3d are shown to estimate the sum of 4 different plasma protein components possessing C3d but not C3c epitopes. All 4 components were C3-derived polypeptides as shown by activating serum containing 125I-labelled C3, isolating the anti-C3d reactive material in 14% PEG supernatant, followed by analysis on SDS-PAGE and autoradiography. Identical results were obtained by radiolabelling 14% PEG plasma supernatants followed by analysis of the anti-C3d reactive material. The components are referred to as d1, d1', d2 and d3 based on their relative electrophoretic mobilities (alpha 1, alpha 1, alpha 2 and alpha 2 respectively) judged by crossed immunoelectrophoresis. Their apparent molecular weights by SDS-PAGE were 129K (d1), 110K (d1'), 46K (d3) and 45K (d2). The possibility that one or more of the C3d containing components represented a complex of a C3 fragment with another plasma protein was investigated. The role of these components in the scheme of the physiological breakdown of C3 and the importance of the individual C3d components as indicators of complement activation in clinical materials is discussed. It is proposed that the 45K d2 component represents a final physiological breakdown product of C3 in human serum.     

IN VITRO STUDIES OF COMPLEMENT FUNCTION IN SERA OF C4-DEFICIENT GUINEA PIGS

In vitro studies were performed utilizing sera from a strain of guinea pigs with a total absence of hemolytically active C4. Previous studies in these animals have demonstrated normal complement-dependent inflammatory reactions, suggesting that they are able to bypass their deficiency of C4. In vitro studies with C4-deficient serum also indicate normal activation of late-acting C components. Thus, endotoxin was capable of fixing normal amounts of the late components of complement (C3-9) in these sera, but did not fix C1 and C2. Antigen-antibody complexes fixed both early and late components of complement, although components beyond C4 were fixed less efficiently than in normal sera. Therefore, both in vivo and in vitro evidence indicates that the C4-deficient guinea pigs possess an alternate pathway for activation of late-acting complement components. Antigenic analysis of C4-deficient serum utilizing both guinea pig anti-C4 antibody and rabbit anti-C4 antibody suggests an absolute deficiency of C4-like molecules. Sera from animals with C4-deficiency were found to have one-half the normal level of C2. Sera from five of eight animals tested had 10–20% normal C1 activity. C3-9 assayed as a complex was normal.     

Reproducible in vitro preparation of intermediate C3d coated red blood cells

As a standardizing reagent and/or as a positive control for the anti- C3d reactivity of antiglobulin reagents, test red blood cells (RBC) reproducibly coated with "C3d only" (i.e. lacking other complement components and immunoglobulins) are essential. We have prepared RBC coated by intermediate amounts of C3d. Two approaches to varying the amount of C3d bound to RBC were studied: a) variation in Mg++ concentration and b) dilution of donor plasma. The amount of C3d bound to RBC was assessed both by agglutination reactions with serial dilutions of a standard anti-C3d serum and by quantitation of bound anti-C3d with 125I-labeled anti-antiglobulin serum. Marked individual donor differences were encountered in response to varying Mg++ concentration and to dilution of donor plasma; no single set of conditions could be employed to produce a desired intermediate C3d- coated RBC from all donors. Examples of variations to be expected with both manipulations are illustrated, along with studies of conditions under which the standard deviation for bound C3d on intermediate C3d- coated RBC made from 5-donor pools was less than 10%.     

Antigenic determinants of C3 and C4 complement components on washed erythrocytes from normal persons

Well-washed erythrocytes from normal persons were agglutinated by antisera to C3, C3d, and C4, and this agglutination was specifically inhibited by the corresponding C3 or C4 protein. C3 and C4 antigenic determinants were present on the red blood cells of freshly shed blood promptly anticoagulated with EDTA, heparin, ACD, or CPD, and no significant changes in degree of agglutination were observed on storage of EDTA or CPD blood for two weeks at 4 C. Marked differences in degree of agglutination by anti-C3, anti-C3d, and anti-C4 were observed when erythrocytes of 16 normal persons were assayed, and significant correlations were obtained when the quantitative results with any two antisera were compared. Anti-C3c did not agglutinate erythrocytes from normal persons, suggesting that the C3 antigens detected on normal cells are carried by the C3d fragment. To avoid significant agglutination of the erythrocytes from some normal persons, very dilute preparations of anti-C3, -C3d, and -C4 had to be used for instrumented diagnostic direct antiglobulin tests. Stronger reagents could be used for indirect antiglobulin tests when the result of a suitable control could be subtracted.     

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.     

Quantitation of the third component of complement on stored red cells

By means of an automated anti-C3c consumption technic, we quantitated the molecules of the third component of human complement (containing the C3c fragment) that accumulate on red cells (RBCs) during liquid whole-blood storage. Calibrated C-3 sensitized zymosan particles ( ZyC3 ) were used as standards for bound C3. Although the dose-response curves of anti-C3c neutralization by ZyC3 and stored RBCs were similar, mathematical analysis showed that the shapes of these two sigmoidal curves were significantly different. This indicated that the large C3 molecules bound to stored RBCs differed antigenically from those bound to ZyC3. Despite this difference, the former could be quantitated adequately by the automated anti-C3c consumption technic. Red cell- bound large C3 molecules were measured following various periods of storage at 4 degrees C of whole blood samples (n=102). An average of 48 molecules were detected on RBCs stored for 21 days. Statistical analysis of these data indicated that during storage at 4 degrees C there was a continuous accumulation of C3 on RBCs, followed by cleavage of C3c fragments. The degree of agglutination of stored RBCs with anti- C3c was proportional to the number of cell-bound large C3 molecules.     

Monoclonal antibodies in blood group serology

The state of the art of monoclonal antibody specifications and blending characteristics needed to produce high quality reagents are described for ABO, RhD and polyspecific anti-human globulin (AHG) reagents.Some interesting advances have been made. Thus some murine monoclonal anti-As (e.g. MH04) see Ax. Also, they may show feeble reactions with the traces of A on some group B cells (the B(A) phenomenon) and the quality control of these reagents is discussed. The use of anti-A,B reagents is now debatable as monoclonal anti-A/anti-B reagents are now more reliable than conventional reagents for detecting weak sub-groups of A and B.RhD typing by saline routine and rapid tests can now be performed with IgM monoclonal anti-D reagents (e.g. MAD-2), but the old problem of Du and D variants is discussed as IgM anti-D is not reliable for their detection.Monoclonal anti-C3c/C3d or just selected anti-C3d (BRIC-8) blended with rabbit anti-IgG make excellent potent clean AHG reagents. The essential false positive test of fresh serum incubated with CPD-A1 red cells from donor pack segment lines is essential for the adjustment and evaluation of AHG reagents.Selected monoclonal anti-M, anti-N, anti-Le^a and Le^b are established as excellent typing reagents and monoclonal antibodies have now been developed to e, E, K, k and other red cell antigens.     

A trypsin method for coating human red blood cells with plasma C3 and/or C4

Simple procedures are described for the production of human red blood cells coated with C3 and/or C4 by brief trypsin treatment of cell- plasma mixtures at room temperature. These cells ('EC3/4'-try) were unusual in that their agglutinability by anticomplement sera, as compared to sucrose-water RBC, was similar when tested by AutoAnalyzer but was markedly reduced in manual testing, especially against anti-C4. In spite of this, both 'EC34'-try and 'EC3'-try were found to be suitable indicator cells for determining the adequacy of anti-C3c and/or anti-C3d activities in reagents used for manual antiglobulin tests.     

Epitope specificities and quantitative and serologic aspects of monoclonal complement (C3c and C3d) antibodies

Twenty-two monoclonal antibodies to human C3c and ten to C3d were obtained by hybridization after the immunization of mice with complement-coated human red cells and/or purified human complement components. C3c antibodies were variable in their agglutination reactions with cells coated with C3 by antibody in vitro; more consistent and potent reactions with these cells were observed with anti-C3d, and all anti-C3d reacted with red cells coated with C3 in vivo. Immunoradiometric assays were used to estimate antibody concentration, affinity, and epitope specificity. The antibody content in ascitic fluids varied from less than 0.1 mg per ml to 5.6 mg per ml. The estimated values of antibody affinities for Sepharose-coupled C3 ranged from 2.8 X 10(6) l per M to 5.0 X 10(8) l per M; on average, IgM antibodies had higher affinities than IgG antibodies. Competitive binding assays showed that the monoclonal antibodies recognized at least seven different epitopes, four on the C3c and three on the C3d fragment of C3. When the results of serologic and quantitative assays were compared, no convincing relationship was found between serologic performance and epitope specificity, antibody concentration, or affinity. IgM antibodies generally gave higher agglutination scores than IgG antibodies, and Ig class was the only useful predictor of serologic efficacy.     

Serological and histological features of anti-HCV-positive individuals without serum HCV RNA.

Antibody to hepatitis C virus (anti-HCV) in patients who are negative for HCV RNA in serum may indicate a memory of past infection of HCV. However, their clinical features have not been well understood. Fourteen anti-HCV-positive but HCV RNA-negative individuals were examined for serological and histological features. As a result, it was found that they had only antibody to HCV core antigen C22-3 with or without antibody to nonstructural viral antigen C33c on a recombinant immunoblot assay (RIBA), and that an concentration of anti-C22 was low. Liver biopsy showed two having no evidence of an obvious hepatic injury, two having a minimal change, and two having portal fibrosis. HCV RNA was not found in the liver. These results corroborate an idea that the anti-HCV in HCV RNA-negative individuals implies a past infection of HCV. Furthermore, it is suggested that a combination of an appearance pattern of antibody to HCV antigens on RIBA and anti-C22 titer are an useful marker to distinguish anti-HCV-positive individuals without viremia from those with viremia.     

Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes

Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab’)(2) anti-CR(1) (C3b receptor) or F(ab’)(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab’)(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab’)(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation-linked acquisition of the abiity to secrete elastase that cleaved reagent particle-bound C3bi into CR(3)-unreactive C3d. Neither neutrophils nor monocytes bound C3d-coated particles at any stage of maturation. Assay of CR(3) with mature neutrophils required inhibition of neutrophil elastase with either soybean trypsin inhibitor or anti-elastase antibodies, and the amounts of these elastase inhibitors required to allow EC3bi rosette formation increased with neutrophil maturation. Because lymphocytes bound C3bi to CR(2) as well as to CR(3), specific assay of lymphocyte CR(3) required saturation of membrane CR(2) with Fab’ anti-CR(2) before assay for rosettes with C3bi-ms. Only 3.5 percent of anti-CR(2)- treated peripheral blood lymphocytes bound C3bi-ms. Therefore, among normal blood lymphocytes the majority of the 12 percent C3bi-ms-binding cells expressed only CR(2) (8.5 percent), and the small proportion of C3bi-ms- binding cells that expressed CR(3) (3.5 percent) represented a distinct subset from the CR2(+) cells. Double-label assay indicated that 3.0 percent out of 3.5 percent of these CR(3)-bearing lymphocytes were B cells because they expressed membrane immunoglobulins. Of the remaining CR(3)(+) cells, 0.2 percent expressed either Leu-1 or 3A1 T cell antigens, and 0.6 percent expressed the OKM-1 monocyte-null lymphocyte determinant.     

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.