Non-complement Fixing Antibodies Research Articles

Improved flow cytometry crossmatching in kidney transplantation.

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20+ B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.

P114 : CHANGE OF THE PATIENT’S ANTI HLA ANTIBODY PROFILE BETWEEN INITIAL AND FINAL WORK-UP

Aim Patients anti HLA antibody profile could change overtime in its specificity, strength and/or behavior. Here we are presenting a case for a patient with end stage renal disease (ESRD) workup for living related kidney transplant, in which antibody profile changed between the initial and final work-up (the day before transplant). Methods HLA Class I highly sensitized patient with anti A26 and anti B50 Donor Specific Antibody (DSA) with 2904, 12168 Median fluorescent Intensity (MFI) respectively, underwent desensitization protocol with Rituximab, high dose IVIG and Plasma Apheresis. One day before transplantation, final Complement Dependent Cytotoxicity crossmatch (CDCXM) as well as Flow Cytometry Crossmatch (FCXM) were performed utilizing historical serum “initial work-up sera prior to desensitization” and current serum post desensitization. Results As can be seen in the table below, the characteristics of the patients’ DSA changed between the initial and final work-up. The DSA changed from complement fixing antibody with a positive CDCXM crossmatch to non-complement fixing antibodies with negative CDCXM crossmatch. Conclusion This case highlights the relevant of frequent testing of anti-HLA antibodies in renal transplant candidates. As seen above, the antibody Isotype switched from fixing to none complement fixing. Thereafter, such conversion could modify the treatment modality. Result. Initial work-up Final work-up CDCXM Positive Negative FCXM Positive Positive Undiluted sera A26 (2904 MFI), B50 (12168 MFI) A26 (12421 MFI), B50 (984 MFI) Diluted sera (1:4) A26 (16680 MFI), B50 (6247 MFI) A26 (5936 MFI), B50 (Negative) C1q Positive Negative

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Aim The method of choice in most of the HLA laboratories for the identification of anti HLA antibodies is the solid phase assay Luminex technology. This method is very sensitive and it is being used for virtual crossmatching as well as for paired kidney exchange (PKE) programs. The most challenging factor for the paired kidney exchange programs is the high sensitization level of the listed couples determined by Luminex-IgG MFI. Now it is increasingly believed that the complement fixing donor specific antibodies (DSA) are the more clinically relevant than non-complement fixing antibodies. In this study we determined impact of using Luminex C1q instead of Luminex-IgG in the pool of the couples listed in our PKE program. Methods A cohort of 12 patients was chosen randomly from the total of 28 couples constituting our HLA incompatible PKE program. All samples were tested by Luminex-IgG and Luminex-C1q (One Lambda) for HLA class I and II antibodies. The unacceptable antigens listed in the PKE are those with more than 6000 MFI for HLA-A,B,DR and DQ. For HLA-C it was 10000 MFI. Results The percentage of complement fixing antibodies detected for HLA class I and II were 36% and 35% respectively. When the unacceptable antigens entered in the PKE program were those Luminex-IgG positive, there were no exchangeable pairs. However when the unacceptable antigens entered in the PKE program were only those C1q positive, we found four one-way exchangeable pairs. Conclusions Our data suggest that we should consider listing unacceptable antigens in the PKE program using the Luminex-C1q positive instead of Luminex-IgG positive. Listing unacceptable antigen in the PKE program solely based on Luminex-IgG will deprive many patients from being transplanted with compatible pairs.

Clinical Significance of a Positive Cross Match in the Setting of Non-Complement Fixing Donor Specific Antibodies in Adult Heart Transplant Recipients

Purpose The presence of donor specific antibodies (DSAs) increases the risk of antibody mediated rejection (AMR) and coronary vasculopathy in heart transplant recipients. Identifying non complement binding DSAs may allow successful transplantation of highly sensitized patients. We analyzed clinical outcomes of adult heart transplant recipients with a positive flow cytometric cross match (FXM) but non complement fixing antibodies as determined by the C1q assay. Methods and Materials Charts of transplant recipients at our institution were reviewed from January 2009 to July 2012 to identify patients with DSAs and a positive FXM and to analyze outcomes. Results Seventy patients were transplanted during this time; five had a positive prospective FXM followed by transplant if their DSA mean fluorescent intensity (MFI) was figure 1 ] Conclusions Differentiating complement and non-complement fixing antibodies using the C1q assay may help in the risk assessment of transplanting a patient with a positive FXM. PreTx PRA (class I/II) # of DSAs # of rejected donors FXM (T/B) AMR Death 1 47/0 1 2 +/- 0 0 2 0/96 2 11 -/+ 0 0 3 100/45 1 1 +/+ n/a YES 4 67/9 2 13 +/+ 0 0 5 98/72 2 12 -/+ 0 0

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Aim It is widely believed that complement fixing donor specific antibody is more clinically relevant than non-complement fixing antibody. However, the C1q assay, which is used to determine if there is complement fixing Donor Specific Antibody (DSA) is time consuming and expensive. We wanted to study if the Luminex Single Antigen (SA) MFI level is related to the presence of C1q fixing antibody. Methods Twelve kidney recipient sera, demonstrating a total of 29 anti-HLA DSAs by the SA assay (One Lambda, Canoga Park, CA) were tested by the C1q assay (One Lambda, Canoga Park, CA). Results Five of these sera demonstrated C1q fixing DSAs. The average MFI of the SA for these positive sera was 17,920. The average MFI of the SA for the sera not having C1q antibody specificities was 4776. There were three sera that had relatively high MFI values that were not positive by the C1q assay (MFI average 12,269) [Fig. 1]. Download full-size image Conclusions The strength of the MFI in classical Luminex SA testing has a high predictive value for positivity in the C1q assay. We found that sera with greater than 13,000 MFI by Luminex SA had a high probability of being positive by the C1q assay. Therefore, the use of the classical Luminex SA assay to determine the strength of the MFI is beneficial to determine if reflex C1q testing is warranted.

Review article: Luminex technology for HLA antibody detection in organ transplantation

Since its inception in the early 1960s, the serologically based complement-dependent cytotoxicity (CDC) assay has been the cornerstone technique for the detection of human leucocyte antigen (HLA) antibodies, not only in pre-transplant renal patients, but also in other forms of organ transplantation. Recently, solid phase assays have been developed and introduced for this purpose, and in particular the Flow-based bead assays such as the Luminex system. This latter assay has proved to be far more sensitive than the CDC assay and has revealed pre-sensitization in potential transplant recipients not detected by other methods of HLA antibody detection. However, the clinical implications of this increased sensitivity have not been convincingly demonstrated until recently. This technology for HLA antibody detection permits the evaluation of the clinical importance of antibodies directed at, for example, HLA-DPB1 and HLA-DQA1, which has not been possible to date. There are Luminex issues, however, requiring resolution such as the ability to distinguish between complement fixing and non-complement fixing antibodies and determination of their relative clinical significance. Luminex technology will permit a re-evaluation of the role of HLA antibodies in both early and late antibody-mediated rejection.

Humoral Responses Directed Against Non-Human Leukocyte Antigens in Solid-Organ Transplantation

Antibody-mediated mechanisms have a major impact on allograft function and survival. During the last decade, improved immunohistochemical and serologic diagnostic procedures have been developed to monitor antibody responses against human leukocyte antigens (HLA). Acute and chronic allograft rejection can occur in HLA-identical sibling transplants implicating the importance of immune response against non-HLA targets. Non-HLA, complement and noncomplement-fixing antibodies may be responsible for a variety of allograft injuries, reflecting the complexity of their acute and chronic actions. Non-HLA antibodies may occur as alloantibodies or autoantibodies. Their antigenic targets described, thus, far include various minor histocompatibility antigens, vascular receptors, adhesion molecules, and intermediate filaments. An analysis of the subtle mechanistic differences in the individual antibody responses directed against non-HLA may help to identify patients at particular risk for irreversible acute or chronic allograft injuries and improve overall outcomes. This review summarizes the current state of research, development in diagnostic and therapeutic strategies, discusses some emerging problems, and provides perspectives in the area of humoral response against non-HLA in solid-organ transplantation.

Anti‐Gal antibody‐mediated skin graft rejection requires a threshold level of Gal expression

Despite overcoming xenograft hyperacute rejection (HAR), Gal (galactose-alpha1,3-galactose) expression may not be completely eliminated from the alpha1,3-galactosyltransferase gene knockout (Gal KO) pig because of alternative galactosyltransferases. Whether low levels of "residual" Gal are still susceptible to either complement fixing or non-complement fixing antibody beyond the HAR barrier remains unknown. Furthermore, it would be impossible to analyze the immune response specific to low-level Gal in a xenograft setting given the multitude of xenoantigens that could induce a recipient response. To investigate this question, we therefore used a skin graft model in BALB/c mice where the sole difference between donor and recipient was the expression of Gal, where rejection is caused by passively administered anti-Gal monoclonal antibody and where HAR does not occur. Gal expression over time was examined by immunohistochemistry in wildtype-to-Gal KO skin grafts. Graft rejection in response to passively administered anti-Gal monoclonal antibody at early and late time points was studied to determine changes in susceptibility to antibody. To independently test the effect of reduced Gal expression on antibody-mediated rejection, we used two separate lines of alpha1,2-fucosyltransferase transgenic mice as skin donors in the model. These mice have known reduced but different levels of Gal as determined by flow cytometry on peripheral blood leukocytes. Gal expression on skin grafts diminished with time with a corresponding reduction in susceptibility to antibody-mediated rejection. Skin grafts at day 30 (n = 7) and 150 (n = 11) had a rejection rate of 100% and 45% respectively in response to non-complement fixing anti-Gal antibody administered to the recipient. Similar results were demonstrated with a complement fixing anti-Gal antibody. When alpha1,2-fucosyltransferase transgenic mice skin was used in the model, the line with lowest level of Gal expression was resistant to antibody-induced rejection with a rate 0% (n = 9) vs. 60% (n = 5) in the alternative line with relatively more Gal expressed but still much less than normal mice. Resistance to anti-Gal antibody-mediated damage in the model was observed in skin grafts 100 to 150 days post-grafting but not earlier and was associated with a reduction in Gal expression. It is possible that below a threshold level of Gal expression, the grafts were not susceptible to anti-Gal antibody.

11: C4D Fixing, Luminex Binding Antibodies – A New Tool for Prediction of Graft Failure after Heart Transplantation

It is well established that detection of pre-formed donor specific HLA antibodies by the complement dependent cytotoxic (CDC) test leads to accelerated graft failure. The introduction of sensitive solid phase assays, which do not distinguish between complement fixing and non-complement fixing antibodies, has led to an increase in numbers of antibody-positive patients, without a clear indication of when it is safe to transplant. The purpose of this study was to analyse Luminex detected antibodies, their ability to fix C4d and to determine the effect on cardiac allograft survival.

Functional analysis of schistosomes EF-hand domain-containing tegument proteins

Schistosomes cause schistosomiasis disease which severely threatens human health. Little is known about the functions of EF-hand domain containing schistosomes tegument proteins other than as antigens. More possible functions of these tegument proteins were investigated with in silico analyses including protein-protein functional interaction, site-specific variation and glycosylation modification. The analysis results suggested that schistosomes could actively modulate host immune responses for its own favor through functional interactions with host proteins with immunomodulatory function, and passively regulate host immune responses through sequence variation under positive selection and glycosylating the recognition sites of host immune attack. In addition, the analysis of the C-terminal domain of these tegument proteins indicated that they could assist schistosomes in escaping host immune attacks through inhibiting chemotaxis and non-complement fixing antibody (IgG4) responses. In summary, our results suggested that these tegument antigen proteins could assist schistosomes in escaping and modulating host immune responses for self-protection during the process of host-parasite interaction.

Bacillus subtilis spores: A novel microparticle adjuvant which can instruct a balanced Th1 and Th2 immune response to specific antigen

There is a current need for safe, cheap, and effective vaccine adjuvants, to combine with sub-unit antigens to enhance their immunogenicity. In this study we have used probiotic Bacillus subtilis spores, known to be safe and fully tolerated by ingestion in man, and explored their ability to influence the magnitude and diversity of immune responses induced against two model antigens, tetanus toxoid fragment C (TT) and ovalbumin (OVA) in mice. The results show that B. subtilis spores not only increased antibody and T cell responses to a co-administered soluble antigen, but also broadened them, to include both antigen-specific CD4+ and CD8+ T cell responses as well as complement and non-complement fixing antibody isotypes. Furthermore, following intranasal immunization, spores augmented specific IgA to co-administered antigen both in the local respiratory and distal vaginal mucosa, as well as increased antigen-specific IgG antibody in draining LN and blood. Collectively, these data demonstrate that naturally occurring, non-pathogenic, non-commensal spores of B. subtilis both instruct and augment polyvalent immune responses and highlight their clinical potential in future vaccines to generate broad-based immunity.

Screening of an Echinococcus granulosus cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental protein involved in the immune escape.

The worldwide problem of chronic Echinococcus granulosus disease calls for new parasite-derived immunomodulatory molecules. By screening an E. granulosus cDNA library with IgG4 from patients with active cystic echinococcosis, we identified a cDNA that encodes a predicted partial protein that immunofluorescence studies localized in the protoscolex tegument and on the germinal layer of cyst wall. We named this protein EgTeg because the 105 amino acid sequence scored highest against a family of Schistosoma tegumental proteins. Evaluating the role of EgTeg in the human early inflammatory response we found that EgTeg significantly inhibited polymorphonuclear cell (PMN) chemotaxis. Cytometric analysis of intracellular cytokines disclosed a significantly higher percentage of cells producing IL-4 than IFN-gamma (P = 0.001, Student's t-test) in T lymphocytes from patients with cystic echinococcosis stimulated with EgTeg. EgTeg induced weak Th1-dependent proliferation in 42% of patients' peripheral blood mononuclear cells. In immunoblotting (IB) analysis of total IgG and IgG subclass responses to EgTeg in patients with cystic echinococcosis, patients with other parasitoses, patients with cystic lesions and healthy controls, total IgG specific to EgTeg yielded high sensitivity (73%) but low specificity (44%) precluding its use in immunodiagnosis. Conversely, IgG4 specific to EgTeg gave acceptable sensitivity (65%) and high specificity (89%) suggesting its use in immunodiagnosis to confirm ultrasound documented cysts suggestive of E. granulosus. Because the new tegumental antigen EgTeg inhibits chemotaxis, induces IL-4-positive T lymphocytes and noncomplement fixing antibodies (IgG4) it is an immunomodulatory molecule associated with chronic infection.

Antibody deposition in the stria vascularis of the MRL-Fas lpr mouse

The MRL-Fas lpr mouse, a model of multisystemic, organ non-specific autoimmune disease, has been proposed as a model of immune-mediated inner ear disease. Preliminary studies indicate that it develops cochlear pathology focused in the stria vascularis including intracellular edema and degeneration which develops in the absence of an inflammatory infiltrate but in the presence of antibody deposition. It was thus hypothesized that the antibodies found in the stria were mediating a direct pathologic effect on this structure, without recruiting classical inflammatory mediators. It was further hypothesized that the antibodies deposited within the stria would be derived from the non-complement fixing isotypes and subclasses, which are known to be able to mediate direct pathologic effects on target tissues. This study utilized immunohistologic techniques to identify the antibody isotypes and subclasses deposited within the stria vascularis of the MRL-Fas lpr mouse. Results indicate that all antibody isotypes and subclasses can be identified within the stria vascularis in the absence of complement. Thus, antibody deposition was not restricted to non-complement fixing antibodies. While it is possible that antibodies are mediating direct pathologic effects within the stria, the non-specific nature of the antibody deposition may indicate that these antibodies are not responsible for the observed pathology. Rather, other mechanisms, such as metabolic and genetic etiologies, must also be considered.

Pretransplant crossmatching: an individualized process

I N THE mid-1960s Terasaki introduced the complement dependent cytotoxicity test (CDC) into clinical transplantation, after positive crossmatch was reported to be strongly predictive of hypcracute kidney rejections.’ At prcscnt it is used to evaluate pretransplant sensitization. The CDC test only detects complement fixing antibodies; undctccted arc noncomplement fixing antibodies that can also be prcscnt in the sera and cause cell damage by antibody dependent cell mediated cytotoxicity.‘,.’ For these antibodies, the anti-human globulin (AHG) test and the flow cytometry (FC) crossmatch wcrc introduced into clinical setups.‘,’ Cellular presensitization might also cause immediate immunological injury of the graft.” However, no cellular test is currently being used to measure sensitization in clinical transplantation. Resulting from a shortage of cadaveric organs, transplantation from living related donors has become a common procedure. Some recipients had already become prescnsitizcd because of blood transfusions, childbirth, and/or previous transplantation. It is therefore imperative to evaluate the immunological status of the recipient. thus avoiding posttransplant immunological graft loss due to donor specific immunization, and not deny organ donations to paticnts with clinically irrelevant antibodies because only anti-HLA antibodies have been found to bc harmful.’ The three kidney transplanted patients presented in this article have case histories in which these aspects were of major clinical significance.

Flow cytometry crossmatching: The first 10 years

In this review, we have briefly summarized the experience with the FCXM in the past 10 years. Amid confusing outcome data early on, a clearer picture is emerging by understanding the nature of the antibodies detected by the FCXM and defining their cellular targets. Numerous studies, especially the most recent, show significant survival differences between FCXM-positive and -negative first transplants, even though a few early studies did not. Moreover, in all studies the FCXM result has consistently shown itself to be important in the case of regrafts and sensitized recipients (Table 4). The variability of the survival data shows that the FCXM is by no means a perfect, or even well-understood, test at this time. To improve its usefulness, we need to define further the roles of the anti-donor antibodies that the FCXM detects: low-titer anti-donor antibodies and noncomplement-fixing antibodies. Furthermore, we need to define even more specifically the targets to which harmful antibodies bind. Perhaps equally important questions to ask are why certain anti-donor antibodies are not harmful to the donor graft and what factors determine their production. It is clear that the FCXM can detect transplants with potentially poor outcomes that the standard CXM misses, but at the cost of false positives. Our task in the next 10 years will be to improve on the sensitivity and specificity of the FCXM, and to integrate the FCXM into the changes in pretransplant and posttransplant immunologic monitoring. A shift in thinking should ensue about how best to use the results of crossmatches and other tests in the pretransplant workup. We should no longer look at these tests individually, but rather integrate their results as a whole into a cohesive cumulative transplant risk formula that will guide us in assessing the potential risk of any given donor-recipient pair. Finally, the role of FCXM in living-related transplants is currently undefined and deserves further careful evaluation.

2 The normal fetomaternal immune relationship

The antigenic status of the preimplantation embryo is ill-defined and there are no clearly recognized maternal immune reactions against this early stage of development. Following implantation, the pregnant female shows evidence of immune recognition of her intrauterine allogeneic conceptus. In a proportion of pregnancies, particularly in multiparous women, there are maternal cytotoxic antibodies exhibiting specificity for the paternally inherited HLA antigens of the fetus. When these are undetectable there may be other antibodies that are non-complement fixing and non-cytotoxic or antibodies that are not present as free molecules and incapable of identification in conventional assays. Anti-HLA antibodies pose no threat to the fetus, principally owing to their absorption by the placenta and, very likely, the harmless binding of any that do reach the fetal circulation. No potentially deleterious cytotoxic T lymphocyte generation occurs in most pregnancies. The extent to which this is due to maternal immunoregulatory control processes is not yet established. The fetal trophoblast is able to act as a protective barrier by virtue of unique properties, including a lack of conventional class I and class II HLA molecules, that render it insusceptible to immune attack. The nature and significance of any maternal recognition of non-HLA antigens on trophoblast await elucidation. Maternal immune cell traffic across the placenta occurs only at a very low level, if at all, in normal pregnancy. This may take place to a greater degree in some of the rare instances of fetal graft-versus-host disease, but this is complicated by the associated fetal immunodeficiency. Maternal IgG antibodies are transmitted across the placental trophoblast by receptor-dependent mechanisms to provide immediate protection for the neonate against environmental pathogens. Leakage of fetal erythrocytes, leukocytes and platelets into the maternal circulation can elicit IgG isoantibodies that take advantage of the same mechanisms to gain access to the fetus, with pathological consequences. Autoantibodies in women with various disease states may similarly pass into the fetus but these normally produce only mild and transient effects. The development of the fetal immune system begins at an early stage of gestation. It is competent to respond to intrauterine infections from as early as 12 weeks and has full functional potential at birth. Maternally acquired IgG is available for up to 9 months of life until the infant's own immune system has been adequately primed and activated following first exposure to specific antigens. The normal fetomaternal immune relationship represents a remarkable harmonious association between two genetically disparate individuals.(ABSTRACT TRUNCATED AT 400 WORDS)

IgG Subclasses of Intercellular and Basement Membrane Zone Antibodies: The Relationship to the Capability of Complement Fixation

There are four main subclasses of human IgG: IgG1, IgG2, IgG3, and IgG4, among which IgG1-IgG3 activate complement, but IgG4 does not. We studied the IgG subclasses of anti-intercellular (IC) antibodies in pemphigus patients and anti-basement membrane zone (BMZ) antibodies in bullous pemphigoid (BP) patients by immunofluorescent staining using mouse monoclonal antibodies against human IgG1-IgG4. At the same time, the capability of complement fixation of each serum was determined by means of complement immunofluorescence. In both pemphigus and BP autoantibodies, various distributions of IgG subclass were shown, but specific patterns were not observed. In BP, all of the complement fixing antibodies had at least one of IgG1-IgG3 subclasses, while noncomplement fixing antibodies only possessed IgG4. This result agreed well with the biologic characteristics of IgG subclasses in respect of complement fixing capability. On the contrary, in pemphigus, the circulating antibodies showed a distribution of IgG subclass which did not correlate with the biologic characteristics in terms of complement activation. This discrepancy may further dispute the roles of the complement system on the bulla formation in pemphigus.

IgG4 and hyposensitization.

The mechanism of action of hyposensitization is still unclear. Probably, allergen-blocking antibodies have some effect, in particular in insect venom allergy, but it is likely that other mechanisms are operative as well. With the types of treatment currently available suppression of the production of specific IgE antibodies does not seem to be a dominating factor. In view of the presumed minor contribution of allergen-blocking antibodies in inhalant allergy, it is as yet impossible to define the role of IgG4 antibodies other than quantitatively: It is the main allergen-blocking antibody! There is no conclusive evidence that the IgG4 is the exclusive allergen-blocking antibody in immediate type allergy. However, its main biologic significance might well be on a completely different level, viz. prevention of immune complex disease. The production of non-complement fixing antibodies, unable to form large complexes due to functional monovalency, during prolonged antigenic exposure seems to provide adequate protection against complement-induced damage and other sequela of precipitating immune complexes that can be expected from a persistent production of IgG1. The rare occurrence of immune complex induced disorders during hyposensitization therapy is possibly a result of this phenomenon.

Antigenic determinants on myeloid leukaemia colony‐forming cells resemble those of normal myeloid progenitor cells and differ from those of circulating blast cells

We studied the antigenic characteristics of leukaemic colony-forming cells (CFU-L) from the blood of patients with chronic granulocytic leukaemia (CGL) in blastic transformation (BT) and acute myeloid leukaemia (AML) by in vitro culture techniques after complement-mediated lysis with one anti-DR and 10 selected myeloid monoclonal antibodies (McAbs), all of which were cytotoxic in the presence of complement. At the same time we studied the antigenic characteristics of the circulating blast cells from the same patients using in addition one non-complement fixing antibody (BI.3C5) with standard immunofluorescence and immunoalkaline phosphatase techniques. We also used myeloid progenitor cell assays in conjunction with cytotoxic McAbs to investigate the antigenic determinants on Day 7 CFU-GM, Day 14 CFU-GM and BFU-E from the blood of patients with CGL in chronic phase (CP) and from normal bone marrow. We found that two of the McAbs, S4-7 and WGHS29.1, recognized a higher proportion of CFU-L from the blood of AML patients than from patients with CGL-BT. However, the patterns of reactivity for CFU-L from CGL-BT and AML patients with the other McAbs quite closely resembled those observed in CFU-GM and BFU-E from normal individuals and patients with CGL in CP. A McAb with DR specificity and one of the myeloid McAbs, 54/39, recognized both CFU-L from CGL-BT and AML and reacted also with circulating blast cells from the same patients. In contrast, six of the other myeloid McAbs that recognized CFU-L failed to label the corresponding blast cells. We conclude that the antigenic properties of CFU-L in CGL-BT and AML are very similar to, but perhaps not identical with, those of normal CFU-GM and BFU-E. There was a major discrepancy in the antigenic profiles of CFU-L and of the blast cells predominating in the blood.

Critical appraisal of complement dependent microlymphocytotoxicity assay for detecting donor-specific alloantibody pretransplant—importance of indirect immunofluorescence as a superior alternative

The ability of complement-dependent microlymphocytotoxicity assay (CdL) to detect and discriminate between the various types of donor-specific alloantibodies was reevaluated. Data obtained with the CdL assay on purified B and T lymphocytes at warm and cold temperatures was compared to other modes of antibody detection, i.e., indirect immunofluorescence (IF) and the noncomplement-dependent antibody-dependent cellular cytotoxicity (ADCC). Additionally, the significance of antibodies as detected by CdL and IF was ascertained by corelating with kidney transplant outcome. It became apparent that the CdL assay identified weaklyreactive HLA- ABC alloantibodies as being B cell specific. Such weakly reactive HLA-ABC antibodies were also not appreciated in the presence of the cold reactive IgM antibody. Accelerated rejections were the rule in the presence of weakly reactive HLA-ABC alloantibodies indicating that their detection was highly important. The IF assay could discriminate between the antibody class. could detect weakly reactive HLA-ABC alloantibodies, and could detect noncomplement fixing antibodies (ADCC). Further, use of IF prevented us from unnecessarily denying transplants to certain recipients when a positive CdL assay resulted from an IgM antibody or poor cell viability.