Blood Group Molecules Research Articles

Autoantigen presentation by splenic dendritic cells is required for RBC-specific autoimmunity.

Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4+ T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBC-derived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAbfl/fl and Cre-expressing transgenic animals to generate mice that lack I-Ab expression on particular cell subsets. OTII CD4+ T cell proliferation was assessed in vivo in HOD+ I-Abfl/fl xCre+ mice and in vitro upon coculture with sorted APCs. Analysis of HOD+ I-Abfl/fl xCre+ mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4+ T cells. Subsequent in vitro coculture experiments revealed that both CD8+ and CD8- DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.

Investigation of the variable In(Lu) phenotype caused by KLF1 variants.

KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population. In(Lu) samples were sourced through screening and through the RBC reference laboratory. Blood donor samples (8036) were screened to identify weakened/absent Lub antigen. Samples were genotyped by massively parallel sequencing, while surface carbohydrates and blood group molecules were assessed by flow cytometry. Hemoglobin (Hb) types were analyzed by high-performance liquid chromatography. Four of 8036 donors were identified to be In(Lu), and two previously identified In(Lu) samples were provided from the RBC reference laboratory. Five different KLF1 variants were identified; two were novel: c.954G>C/p.Trp318Cys and c.421C>T/p.Arg141*. BCAM and CD44 were reduced in all samples, consistent with previous reports. As a group, In(Lu) RBCs had reduced CD35 (KN), ICAM4 (LW), and CD147 (OK), and demonstrated increased binding of lectins ECA and SNAI. One In(Lu) sample had elevated HbF and another elevated HbA2. Different KLF1 variants may potentially produce variable phenotypes. A framework for investigating KLF1 variants and their phenotypic impact has been provided. In the future, given available international databases, further testing algorithms (as advocated here) will allow for correlation of phenotype with genotype and therefore accurately document this variability between KLF1 variants.

Peripheral but Not Central Tolerance Regulates RBC Autoreactive CD4+ T Cells in a Novel Mouse

Background: Elucidating the mechanisms of T cell tolerance towards autoantigens can be critical for therapeutic efforts to re-establish tolerance in autoimmune diseases or break tolerance in tumor immunotherapy. Tolerance mechanisms have been well described towards tissue-restricted antigens and include deletion, anergy, or regulatory cells; however, T cell tolerance mechanisms against RBC-restricted antigens are poorly understood. Herein, we utilized the HOD mouse to investigate T cell tolerance mechanisms to RBC-specific antigens. The HOD mouse expresses a triple fusion protein consisting of hen egg lysosyme (H EL), ovalbumin (O VA), and human blood group molecule, D uffy (HOD) driven by a RBC-specific promoter. A TCR transgenic mouse that recognizes an OVA peptide presented by MHI II (OT-II mouse) was crossed with the HOD mouse to allow analysis of T cells autoreactive to an RBC specific antigen.Methods: To evaluate central tolerance, OVA specific T cells were analyzed from thymii from 6-8 weeks old OT-II+ HOD+ mice (OT-II+ HOD- littermates were used as controls for T cells in absence of autoantigen). Splenic CD4+ T cells from both OT-II+ HOD+ and control OT-II+ HOD- mice were CFSE-labeled and analyzed 1) in vitro by co-culturing with splenocytes pulsed with whole soluble OVA or 2) in vivo by adoptively transferring into B6.Thy1.1 recipients, followed by transfusion of HOD RBC. Proliferative responses of cells were measured by CFSE dilution analyzed with flow cytometry.Results: There is no detectable autoantibody production against the HOD antigen in OT-II+ HOD+ mice. In the thymus, similar frequencies and total nbers of OVA-specific CD4+ single positive (SP) T cells were observed between OT-II+ HOD+ and OT-II+ HOD- mice. This was not because OT-II+ HOD+ mice were incapable of deleting autoreactive T cells, since OT-II+ HOD+ mice i.v injected with soluble whole OVA, known to induce deletion, had a significant decrease in the CD4+ SP T cells. Phenotypic analysis of mature CD4+ T cells in the spleen revealed fewer OVA-specific CD4+ T cells in OT-II+ HOD+ compared to OT-II+ HOD- mice (p=0.009). The CD4+ T cells present in OT-II+ HOD+ expressed higher levels of markers associated with T cell activation and proliferation such as CD69, CD44, Ki-67 and also decreased levels of L-selection. Moreover, these cells expressed elevated levels of markers associated with T cell anergy and tolerance, including PD-1, CD5 and LAG-3. Finally, OT-II+ HOD+ mice exhibited increased numbers of CD25+ Foxp3+ Tregs (p=0.007). In 2 of 2 experiments (3 mice/group), adoptively transferred CD4+ cells from OT-II+ HOD+ did not proliferate in response to HOD RBCs while OT-II+ HOD- exhibited robust proliferation. In contrast to the in vivo results, CD4+ cells from OT-II+ HOD+ proliferated when co-cultured with splenocytes + whole soluble OVA in vitro.Conclusions: We describe a model in which regulation of CD4+ T cells autoreactive for self-RBC antigen can be studied. Although a significant number of RBC autoreactive T cells are present, and appear to have seen antigen, been activated (i.e. increased CD69 and CD44), and proliferated (increased Ki-67); these cells are nevertheless anergic as indicated by adoptive transfer studies. The ability to proliferate in vitro but not in vivo in response to HOD RBC suggests that removal from the normal in vivo environment removes inhibitory factors, consistent with peripheral tolerance mechanisms in vivo. Indeed, increased Tregs were observed in autoreactive mice compared to control mice. In contrast, thymic deletion seems to play little or no role in this case. Together, these findings identify peripheral and not central tolerance as major control mechanisms of RBC autoreactive CD4+ T cells. Mechanisms may include alterations in expression of PD-1, CD5, and LAG-3 by autoreactive cells. DisclosuresZimring:BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties; Immucor Inc.: Research Funding.

Central T Cell Tolerance and Peripheral B Cell Tolerance for An RBC Autoantigen Are Incomplete in Healthy Mice; Implications for AIHA Pathogenesis

Abstract 693 Introduction:Breakdown of humoral tolerance to red blood cell (RBC) antigens can result in autoimmune hemolytic anemia (AIHA), a severe and potentially fatal disease. The pathogenesis of AIHA is poorly understood. To investigate the baseline biology of tolerance to self-antigens expressed on RBCs, we utilized a murine transgenic mouse with RBC-specific expression of a model antigen consisting of a triple fusion protein of hen egg lysozyme (HEL), ovalbumin (Ova), and human blood group molecule Duffy; HEL-OVA-Duffy (HOD mouse). Methods:Wild-type C57BL/6 (B6) mice or HOD mice (on a B6 background) were immunized with HEL/CFA or OVA/CFA to test immune responses to antigens contained within HOD. Some animals were immunized with peptides as opposed to whole protein. Anti-HOD antibodies were quantified by indirect immunofluorescence using HOD RBCs as targets. Anti-HEL IgG was quantified by ELISA and anti-HEL secreting B cells were enumerated by ELISPOT. CD4+ T cell responses were assessed by tetramer staining and tetramer pull-down assays using I-Ab-OVA-329-337/326-334. T cell tolerance was specifically broken by adoptive transfer of OT-II CD4+ T cells into HOD mice (OT-II T cells recognize OVA323-339 presented by I-Ab). Effects of HOD antigen expression on B cell development were evaluated by crossing the HOD mouse with an anti-HEL BCR knockin mouse (SwHEL mouse) that is capable of normal class switching. Results:Immunization of B6 mice with OVA/CFA induced high titer antibodies reactive with HOD RBCs; in contrast, no anti-HOD was detected in HOD mice immunized with OVA/CFA. Similarly, no anti-HEL was detected in HOD mice immunized with HEL/CFA, whereas wild-type B6 mice had high anti-HEL titers (p<0.05). These data demonstrate overall humoral tolerance to the HOD antigen. Using pull-down assays, OVA-tetramer reactive T cells were detected in both B6 and HOD mice, with similar endogenous frequencies (mean numbers are 40 and 53 T cells, respectively; at least 6 mice analyzed), suggesting that central tolerance did not eliminate HOD reactive T cells. However, upon immunization with OVA peptide, B6 but not HOD mice had a detectable expansion of OVA-tetramer reactive CD4+ T cells, indicating that peripheral tolerance was preventing HOD autoreactive CD4+ T cells from participating in an immune response. To assess B cell tolerance to the HOD antigen, T cell tolerance was circumvented through adoptive transfer or OTII splenocytes (specific for the OVA323-339 peptide) into HOD mice. Anti-HEL autoantibodies were detected in HOD mice but not control B6 mice (p<0.001). Antibody production correlated with a 10–20 fold increase of anti-HEL antibody secreting cells, as determined by ELISPOT. Autoantibody production in HOD mice was not due to passenger B cells from the OTII donor, an artifact of excess CD4+ T cell number, or bystander activation as no autoantibodies were observed upon adoptive transfer with OTIIs on a Rag knockout background, irrelevant CD4+ T cells from SMARTA mice, or activated CD4+ T cells from TCR75 mice. To test the effects of HOD antigen expression on development of autoreactive B cells, HOD mice were crossed with SwHEL BCR transgenic mice (that express anti-HEL) and the F1 mice were analyzed. HEL-reactive B cells were visualized using multimeric HEL conjugated to allophycocyanin. In HOD-SwHEL+ mice, approximately 46±14% of immature bone marrow B cells were reactive with HEL, compared to 15±12% in HOD+SwHEL+ mice (p=0.043, 3 independent experiments, 5 mice total). Conclusions:These data demonstrate that tolerance to an RBC specific antigen is complete in the CD4+ T cell, but not the B cell compartment. CD4+ T cell tolerance appears to be more an effect of peripheral tolerance than central deletion, as OVA-tetramer reactive CD4+ T cells were visible in HOD mice but did not activate upon immunization with their cognate antigen. In contrast, while the HODxSwHEL F1 mice demonstrate that some B cell tolerance to HOD occurs, the induction of autoantibodies by introducing CD4+ autoreactive T cells (OT-II) demonstrates that B cell tolerance to the HOD antigen is incomplete in HOD mice. Together, these data suggest that a breakdown in T cell tolerance is all that is required for the pathogenesis of AIHA. As the T cell tolerance appears not to be deletional, it is predicted that environmental factors leading to a breakdown in peripheral tolerance of CD4+ T cells would be sufficient to induce AIHA. Disclosures:Zimring:Immucor Inc,: Research Funding.

Blood groups: genetics and physiology

Numerous studies in biochemistry, genetics and molecular biology conducted over two decades have shown that blood group gene products expressed on red cells and various tissues exhibit a variety of potential functions, which can be schematically classified into functional groups such as transporters and channels, receptors, adhesion molecules, enzymes or structural components, with a further level of complexity as a same molecule may exhibit multiple functions. As structures present on red cells, blood group and blood group‐related molecules may contribute to the structure and function of the red cell membrane and may help to clarify unresolved biological processes (for instance, water and gas transport through biological membranes). Alternatively, some of these molecules may simply be the witnesses of a residual persistence of membrane components during erythroid cell differentiation with no significant function. Investigation of blood group and blood group‐related molecules as structures present on epithelial or endothelial cells of tissues and organs has revealed other features of their potential physiological role and led to the discovery of some unexpected functions (for instance Fy‐mediated transcytose). Increasing our knowledge on both aspects is of interest not only for transfusion medicine, but also for transplantation and for understanding the normal physiology and physiopathology of several diseases. Current studies indicate that blood group and blood group‐related molecules cover large areas of human physiology from cerebral to renal and reproduction biology. As specific deficiencies of these molecules have only a minor or no detrimental effect in most instances, they are either dispensable for cell function or redundant. In some instances, however, a function becomes apparent under stress or in pathological conditions. How common and rare blood group polymorphisms are maintained and may impact function is largely unknown, although a few examples clearly point to selective pressure exerted by pathogenic micro‐organisms. Despite significant progress, much remains to be discovered to clearly delineate how blood group molecules, alone or as molecular complexes in erythroid and non‐erythroid cells, may act in health and disease, to understand the underlying mechanisms, and, ultimately, how these findings might eventually be translated into clinical applications.

Plasmodium vivax circumsporozoite variants and Duffy blood group genotypes in the Brazilian Amazon region

The circumsporozoite protein (CSP) of the Plasmodium vivax infective sporozoite is considered to be a major target for the development of recombinant malaria vaccines. The Duffy blood group molecule acts as the red blood cell receptor for P. vivax. We review the frequency of P. vivax CSP variants and report their association with the Duffy blood group genotypes from Brazilian Amazon patients carrying P. vivax malaria. Peripheral blood samples were collected from 155 P. vivax-infected individuals from five Brazilian malaria-endemic areas. The P. vivax CSP variants and the Duffy blood group genotypes were assessed using PCR/RFLP. In single infections, the VK210 variant was the commonest followed by the P. vivax-like variant. The typing of P. vivax indicated that the frequency of variants among the study areas was significantly different from one to another. This is the first detection of the VK247 and P. vivax-like variant in single infections in endemic areas of Brazil. Association of the CSP P. vivax variants with the heterozygous Duffy blood group system genotype was significant for VK210 single infection. These observations provide additional data on the Plasmodium-host interactions concerning the Duffy blood group and P. vivax capability of causing human malaria.

Transfusion‐induced autoantibodies and differential immunogenicity of blood group antigens: a novel hypothesis

Blood bank serology has identified hundreds of red blood cell (RBC) antigens contained within numerous blood group systems. Although most blood group antigens are defined by amino acid polymorphisms in the extracellular domain of membrane proteins, it is also possible that additional nonexofacial polymorphisms (NEPs) may exist within cytoplasmic or transmembrane domains. To assess this possibility, we analyzed several blood group molecules by searching the SNPper database for nonsynonymous single-nucleotide polymorphisms. We report the identification of a number of NEPs in the Kell, Kidd, and Duffy molecules. Because the identified NEPs are not exposed on the surface of intact RBCs and are, thus, not accessible to recipient antibodies, they would neither be detected by blood bank serology in vitro, nor would they be recognized targets in hemolytic transfusion reactions in vivo. The presentation of peptides containing NEPs by recipient MHC Class II molecules, however, would nevertheless produce helper T-cell epitopes. In addition to identifying NEPs in human blood group molecules, we explore a novel hypothesis that the presence of NEPs contributes to the immunogenicity of blood group antigens. We further hypothesize that NEPs provide a mechanism by which transfusion can lead to anti-RBC autoantibodies, which are known to occur in humans after transfusion. The scientific basis, existing evidence, approaches to testing, and predicted biology of this hypothesis are presented.

Transcriptional Profile of Carbohydrate Blood Group Genes in Erythroid Versus Neutrophil Differentiation of Human CD34+ Cells In Vitro.

Carbohydrate blood groups and their corresponding antibodies are clinically important, known to be involved in conditions such as hemolytic transfusion reactions, hemolytic disease of the newborn and spontaneous abortion. However, little is understood about the developmental changes in expression of carbohydrate blood groups during hematopoiesis, with preceding studies mainly focusing on protein-based blood group molecules. We have previously identified the carbohydrate blood group A antigen as the earliest, specific marker for definitive erythroid commitment, in preference to other suggested candidates such as Kell or Glycophorin C [Br J Haematol 2004; 127:451–63]. With regard to this lineage-restriction point we mapped the gene expression of some clinically important carbohydrate blood group systems during erythroid versus neutrophil differentiation in vitro. Human bone marrow CD34+ cells from healthy donors, carrying the blood group A1 allele and functional secretor (FUT2) and Lewis (FUT3) genes, were cultured in vitro towards erythroid or neutrophil development and sorted on a flow cytometer into subpopulations according to their surface expression of blood group A antigen/CD117 and CD15/CD33. Sorted cells were cultured in clonogenic assays in methylcellulose or analyzed by TaqMan real-time reverse transcriptase PCR for gene expression of a number of carbohydrate blood group glycosyltransferases. Surface expression of the blood group A antigen coincided with commitment to erythroid differentiation and the expression of CD15 with neutrophil/monocytic differentiation. In gene expression studies the ABO, H (FUT1), I (IGnT) and Pk (A4GALT) genes were all expressed in freshly isolated and sorted CD34+ cells. The ABO and the H genes were up-regulated in erythroid differentiation and silenced in neutrophil differentiation. The ABO gene expression was markedly decreased in late stages of erythroid maturation. The I gene was expressed both during erythroid and neutrophil development with an increased expression in late erythroid differentiation. The Pk gene retained a low expression throughout neutrophil differentiation and was up-regulated several-fold during erythroid differentiation. There was no detectable expression of FUT3 and the gene suggested being responsible for biosynthesis of the Sda antigen, GALGT2, in either erythroid or neutrophil differentiation.Our data support the identification of the blood group A antigen as an early and specific marker for definitive erythroid differentiation. In mature cells of the myeloid lineage, the results of the gene expression studies are compatible with previous findings of gene and/or surface expression of the I and Pk blood groups but not of ABO and H. The marked increase in expression of the Pk gene during erythroid differentiation may well agree with the fact that the Pk antigen is the precursor structure of globoside, the most abundant neutral glycolipid in the erythrocyte membrane. The absence of hematopoietic FUT3 expression in Lewis gene positive individuals was expected whilst the relevance of undetectable GALGT2 expression in hematopoietic differentiation is uncertain. The role of the GALGT2 gene in surface expression of Sda has not been definitively proven and the molecular basis of different Sda phenotype variants is unknown.In conclusion, our data extend previous findings of carbohydrate blood group distribution, primarily obtained from mature blood cells and leukemic cell lines, to normal human hematopoiesis.

Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family

AbstractIdentification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the “Dombrock” blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5′-diphosphate (ADP)–ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Doa versus Dob antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.

Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family

Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the “Dombrock” blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5′-diphosphate (ADP)–ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Doa versus Dob antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.

Time-course expression of polypeptides carrying blood group antigens during human erythroid differentiation.

The time course expression of blood group antigens was examined by flow cytometry using a two-phase liquid culture system that supports the proliferation and maturation of human erythroid progenitors from adult peripheral blood. The progression towards erythroid differentiation was followed by the expression changes of the transferrin receptor (CD71++) and glycophorin A (GPA+). Four main categories of blood group markers were identified: (i) those characterized by an early expression like ABO (A), Kell (K:2) and Rh50 which were detected in the Epo-independent phase 1, (ii) those including GPC (Gerbich, Ge antigens) and Fy6 which were expressed in the late phase 1, (iii) GPA (MN antigens), Wrb (Band 3/GPA interaction), Rh(D, Cc/Ee) and LW which appeared during the Epo-dependent phase 2 and (iv) those like Jk3 and Lub which were expressed in late phase 2. Regarding blood group molecules exhibiting adhesive properties (LW/ICAM-4, Oka and Lu) the most significant event was a sharp decrease of Oka (neurothelin) expression with the concomitant loss of ICAMs expression during the later stage of differentiation. These studies suggest that Oka, ICAMs and LW might contribute to the adhesive interactions involved in the formation of erythroblastic islands and attachment to stroma cells and the extracellular matrix. We also noted an asynchronous expression of the proteins that compose the core of the Rh complex, since Rh50 glycoprotein was expressed earlier than Rh(D, CE) proteins.

ABO glycosyltransferase genotyping by polymerase chain reaction using sequence-specific primers

Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.

ABO Glycosyltransferase Genotyping by Polymerase Chain Reaction Using Sequence-Specific Primers

Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.

Detection of alloantibodies by flow cytometry: relevance to clinical transplantation.

Before an organ transplant is performed, donor-recipient compatibility must be established by a crossmatch in much the same way as it is done for blood transfusions. The target antigens in organ transplantation, however, are HLA rather than blood group molecules, and the target cells are lymphocytes instead of red cells. If antidonor antibodies are detected, it is important to know whether they are IgG or IgM, whether they recognize T and/or B cells, and whether the antibody reactivity is weak or strong. These test requirements are better met by flow cytometry than by the standard cytotoxicity technique. A growing body of evidence now indicates that flow cytometry can provide more sensitive and timely crossmatch information than cytotoxicity assays to decide whether or not a transplant should be done. Flow cytometry crossmatch (FCXM) is a new and evolving technique that has already been found to be extremely useful in the clinical transplantation setting, even though significant questions yet remain about the precision and reliability of using flow cytometry to quantify alloantibodies and about the limits of normal reactivity in the assay. This article reviews important technical details of the FCXM and its interpretation and clinical application in transplantation medicine.