A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

Phenotypic analysis of hematopoietic stem and progenitor cells (HSCs) has been an invaluable tool in defining the biology of stem cell populations. We have recently described the production of AC133, a monoclonal antibody (MoAb) that binds to a novel cell surface antigen present on a CD34bright subset of human HSCs. This antigen is a glycosylated protein with a molecular weight of 120 kD. Here, we report the molecular cloning of a cDNA encoding this antigen and show that it does not share homology with any previously described hematopoietic or other cell surface antigen(s). The AC133 polypeptide has a predicted size of 97 kD and contains five-transmembrane (5-TM) domains with an extracellular N-terminus and a cytoplasmic C-terminus. Whereas the expression of tetraspan (4-TM) and 7-TM molecules is well documented on mature and immature hematopoietic cells and leukocytes, this 5-TM type of structure containing two large (255–amino acid [aa] and 290-aa) extracellular loops is unique and does not share sequence homology with any known multi-TM family members. Expression of this protein appears limited to bone marrow in normal tissue by immunohistochemical staining; however, Northern analysis suggests that the mRNA transcript is present in a variety of tissues such as the kidney, pancreas, placenta, and fetal liver. The AC133 antigen is also expressed on subsets of CD34+ leukemias, suggesting that it may be an important early marker for HSCs, as well as the first described member of a new class of TM receptors.

This study details the profile of 13 cell surface cluster differentiation markers on human reserve stem cells derived from connective tissues. Stem cells were isolated from the connective tissues of dermis and skeletal muscle derived from fetal, mature, and geriatric humans. An insulin/dexamethasone phenotypic bioassay was used to determine the identity of the stem cells from each population. All populations contained lineage-committed myogenic, adipogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineageuncommitted pluripotent stem cells capable of forming muscle, adipocytes, cartilage, bone, fibroblasts, and endothelial cells. Flow cytometric analysis of adult stem cell populations revealed positive staining for CD34 and CD90 and negative staining for CD3, CD4, CD8, CD11c, CD33, CD36, CD38, CD45, CD117, Glycophorin-A, and HLA DR-II. Anat Rec 264: [51][52][53][54][55][56][57][58][59][60][61][62] 2001.

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher orders. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50-70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

OverviewIn renal transplantation, a positive cytotoxic crossmatch between donor cells and recipient serum is associated with early rejection or graft loss and was the driving force behind the establishment of HLA laboratories. Initially, crossmatches were performed by relatively insensitive techniques [e.g. leukoagglutination and direct complement-dependent cytotoxicity (CDC) of target cells]. A negative result justified proceeding, while a positive crossmatch was considered a contraindication to renal transplantation. However, the underlying premises driving this practice, namely that (i) all positive reactions were the result of relevant (i.e. HLA) antibodies that could lead to allograft rejection; and (ii) all negative reactions predicted long-term graft survival were known to be incorrect. From its first clinical description, the simple complementdependent assay was recognized as neither sufficiently specific nor sensitive to identify all relevant antibodies. Over time, more sensitive and specific lymphocyte crossmatch assays were developed that effectively decreased the incidence of early antibody-mediated rejection.In recent years, advances in immunosuppressive therapy have led clinicians to ask whether antibodies identified by these more sophisticated crossmatch techniques represent a contraindication to transplantation. To answer this question, it is essential to prove (or disprove) that antibodies specific for donor HLA antigens are present in recipient sera. A critical analysis of the literature revealed that the majority of studies failed to provide sufficient evidence to ensure that positive (as well as negative) crossmatches were correctly assigned. Indeed, few investigators performed the labor-intensive studies necessary to document that positive crossmatches were the result of antibodies specific for donor HLA antigens. Furthermore, the testing methodology used in those studies was relatively insensitive compared with current and emerging technologies. Given these limitations, we believe it is essential to re-examine the conclusions of studies that formed the basis of our current crossmatch paradigms.Just as the clinical application of calcineurin inhibitors revolutionized transplant medicine, the recent development of HLA antigen specific solid-phase assays (i.e., ELISA and microparticle-based flow cytometric assays) has similarly revolutionized our ability to detect HLA antibodies. Specifically, by documenting whether patient sera possess donor-reactive HLA antibodies, a lymphocyte crossmatch can now be more reliably interpreted. Indeed, with solidphase data, a positive lymphocyte crossmatch can now be categorized as (i) clinically irrelevant, (ii) a risk factor for rejection or graft loss, or (iii) a contraindication to renal transplantation. It is our position that only with accurate risk assessment can desensitization protocols [e.g., intervenous gamma globulin (IVIG) with or without plasmaphereisis] be optimally applied.

In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

Donor-directed human leukocyte antigen (HLA)-specific allo-antibodies (DSAs) cause graft failure in animal models of hematopoietic stem cell transplantation (HCT). Archived pretransplantation sera from graft failure patients (n ‫؍‬ 37) and a matched case-control cohort (n ‫؍‬ 78) were tested to evaluate the role of DSAs in unrelated donor HCT. Controls were matched for disease, disease status, graft type, patient age, and transplantation year.Patients had acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, or myelodysplastic syndrome; 98% received myeloablative conditioning regimens 100% received T-replete grafts, 97% received marrow, 95% HLA-mismatched, and 97% received calcineurin-based graft-versus-host disease prophylaxis. Among the 37 failed transplantations, 9 (24%) recipients possessed DSAs against HLA-A, B, and/or DP, compared with only 1 (1%) of 78 controls. Therefore, the presence of DSAs was significantly associated with graft failure (odds ratio ‫؍‬ 22.84; 95% confidence interval, 3.57-ؕ; P < .001). These results indicate that the presence of pretransplantation DSAs in recipients of unrelated donor HCT is associated with failed engraftment and should be considered in HCT donor selection. (Blood. 2010; 115(13):2704-2708)

Single antigen bead (SAB) testing permits reassessment of immunologic risk for kidney transplantation. Traditionally, high panel reactive antibody (PRA), retransplant and deceased donor (DD) grafts have been associated with increased risk. We hypothesized that this risk was likely mediated by (unrecognized) donor-specific antibody (DSA). We grouped 587 kidney transplants using clinical history and SAB testing of day of transplant serum as 1) unsensitized; PRA=0 (n= 178), 2) 3rd party sensitized; no DSA (n=363), or 3) donor sensitized; with DSA (n=46), and studied rejection rates, death censored graft survival (DCGS), and risk factors for rejection. Antibody-mediated rejection (AMR) rates were increased with DSA (p<0.0001), but not with PRA in the absence of DSA. Cell-mediated rejection (CMR) rates were increased with DSA (p<0.005); with a trend to increased rates when PRA>0 in the absence of DSA (p=0.08). Multivariate analyses showed risk factors for AMR were DSA, worse HLA matching, and female gender; for CMR: DSA, PRA>0 and worse HLA matching. AMR and CMR were associated with decreased DCGS. The presence of DSA is an important predictor of rejection risk, in contrast to traditional risk factors. Further development of immunosuppressive protocols will be facilitated by stratification of rejection risk by donor sensitization.