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Immunogenetic selection of donors for haematopoietic stem cell transplantation: an approach.

Histocompatibility between the patient and his donor is a prerequisite for the success of transplantation of haematopoietic stem cells (HSCT). Histocompatibility testing is clinically performed by HLA typing. HLA typing can be highly informative if performed in families. In this case, comparatively little typing effort often allows to predict the identity of whole HLA haplotypes. HLA typing becomes more demanding, however, if unrelated individuals have to be considered as donors. Given the huge diversity of HLA molecules the answer to the question, whether patient and donor possess 'matched' HLA types requires considerable typing effort. Interaction between the HLA typing laboratory and the clinician is highly needed. It has to be agreed upon what loci are considered relevant and what degree of resolution is necessary. Furthermore, a strategy has to be developed, which ensures that a maximum of potential donors are tested in a reasonable time frame avoiding at the same time a work-overload of the laboratory and an excessive uneconomical typing effort. At present, experience with HSCT with unrelated donors is limited. So there is no consent on the 'correct' way of choosing unrelated donors. Each transplantation centre will have its own characteristic needs, resources, manpower and skills; therefore, the ways vary between centres. I want to present the approach in Vienna.

Alleles↗

Allogeneic bone marrow transplantation with related donors other than HLA MLC-matched siblings, and the use of antithymocyte globulin, prednisone, and methotrexate for prophylaxis of graft-versus-host disease.

Fifteen patients ranging in age from 1-29 years (median age 9 years) had bone marrow transplantations (BMT) from related donors other than HLA mixed lymphocyte culture (MLC) identical siblings. Donors were selected on the basis of HLA similarity and low reactivity in the MLC. Posttransplant immunosuppression consisting of methotrexate (MTX), antithymocyte globulin (ATG), and prednisone was used in an effort to decrease graft-versus-host disease (GVHD). Seven children were treated for aplastic anemia, 7 for hematologic malignancy, and 1 for osteopetrosis. Primary engraftment failure contributed to death in 3 patients, all of whom had aplastic anemia. Nine of 12 engrafted patients developed moderate-to-severe acute graft-versus-host disease. Of the 15 patients, 7 developed interstitial pneumonitis. Three patients demonstrated mixed chimerism (at or beyond 3 months posttransplant). Two of the seven patients treated for aplastic anemia are currently alive six months and more than five years posttransplant; the latter patient has chronic GVHD. Four of the seven patients treated for hematologic malignancy are currently alive more than 500 days posttransplantation. Three have chronic GVHD. Analysis of patient outcome according to the degree of similarity in histocompatibility testing revealed that patients with low reactivity in the MLC (less than 5% relative response in both directions) had a better prognosis (5/6, 83% long-term survival) than patients with maximum donor vs. recipient mixed lymphocyte culture relative response greater than 5% (1/9, 12% long-term survival), P = .016.

Adolescent↗

Progress report on the ASHI/CAP Histocompatibility Survey Program, 1981-1986.

Two histocompatibility testing surveys were conducted over a five-year period by the American Society for Histocompatibility and Immunogenetics and the College of American Pathologists. More than 200 laboratories participated in the HS survey, which consisted of four shipments of cells and serum samples to be tested for ABO type, HLA-A and -B types, antibody identification, and lymphocytotoxicity crossmatching. About 100 laboratories participated in the DR survey, which consisted of four annual shipments to be tested for HLA-DR and -DQ types, antibody identification, and crossmatching. The results of the analysis of the HLA-typing results showed high consistency (generally greater than 90%) among laboratories in the definition of most recognized HLA antigens. In contrast, the HLA antibody screening was generally less consistent between laboratories. On the basis of 90% or greater consensus among participants, it was possible to develop a performance grading system.

ABO Blood-Group System↗

Diagnostic use of molecular probes before and after marrow transplantation.

The application of molecular techniques to disease diagnosis and histocompatibility testing before marrow transplantation and to the evaluation of engraftment and immune reconstitution, and to the identification of infectious and neoplastic complications following transplantation has revolutionized the approach to the care of marrow transplant patients. The techniques of restriction fragment length polymorphism analysis using informative restriction enzymes, the Southern blot technique, and of polymerase chain reaction-amplified DNA and oligonucleotide probe hybridization methods now enable the study of donor and host cells at the nucleotide level.

Amino Acid Sequence↗

The immunogenetics of bone marrow transplantation.

It is now clear that it is not necessary to use an HLA genotypically identical donor to have a successful marrow transplant. However, it is equally clear that the likelihood of complications increases with each increment in histoincompatibility. The implication is that histocompatibility testing must be of the highest possible precision to choose the optimal donor, and to predict the risk of adverse alloreactivity. Most clinicians would seriously consider transplantation from a one locus-mismatched relative or an HLA-matched unrelated donor in virtually any situation in which transplantation from a matched sibling would be felt to be the standard of care. More thought would need to go into transplantation from a two or three locus-mismatched relative or a mismatched unrelated donor.

Bone Marrow Transplantation↗

Unexpected Bw4 and Bw6 reactivity patterns in new alleles.

The Bw4 and Bw6 epitopes were the first HLA-B differences to be recognized by serological methods. Since then 44 serological groups have been identified and more than 250 alleles assigned by molecular typing methods. In general each serological HLA-B group is associated with the presence of either the Bw4 or the Bw6 epitope. There are several exceptions to this rule. Four alleles, B*4601, *7301, *5503 and *1806, show no serological reactivity with either Bw4 or Bw6. Although the Bw6 motif at residues 77-83 is present in these alleles the Bw6 epitope is modified by a valine at residue 76. One or more alleles from the B8, B40 and B62 groups are identified as Bw4 positive, whereas all others are Bw6 positive. In the groups B27, B44 and B47 several alleles are found to be Bw6 positive, while the majority is Bw4 positive. Histocompatibility testing of dialysis patients and their families revealed the serological presence of an unexpected Bw4 epitope associated with B18 in one patient and B56 in another. Allele-specific amplification and sequencing of exons 2 and 3 of these HLA-B alleles revealed the presence of the Bw4 sequence motif for both. The new alleles were assigned B*1809 and B*5607, respectively. In 2 other patients the presence of a new B*07 allele was determined by sequence based typing. Although the new allele, B*0715, showed the Bw6 sequence motif at positions 77 to 83, a substitution of amino acid 76 from glutamic acid to valine was identified. This change resulted in an aberrant Bw6 serological reaction pattern.

Alleles↗

Congenital 21-hydroxylase deficiency as a new deletion mutation. Detection in a proband during subsequent prenatal diagnosis by HLA typing and DNA analysis.

A child with 21-OH-def whose 9 weeks' pregnant mother was referred for prenatal diagnosis was found upon very careful histocompatibility testing to lack expression of any of his father's HLA antigens on his peripheral blood lymphocytes. The possibility of alternative paternity was considered to be extremely unlikely after additional genetic marker tests. The conclusion that the affected child's disease resulted from inheritance of a maternal CYP21B (21-OH) deletion and a de novo deletion in the paternal chromosome 6 segment that includes both the CYP21B (21-OH) and HLA genes was confirmed by subsequent DNA analysis using 21-OH, C4, DPB, and PCH6 probes. The presence of a heterozygous RFLP for DPB, the absence of a deletion for either CYP21B (21-OH) or C4 genes, and the presence of a paternal HLA antigen haplotype on the fetal cells additionally indicated that the fetus lacked the same deletion and could be predicted to be completely normal.

Adrenal Hyperplasia, Congenital↗

Biphasic response of the regional lymphatics in the normal lymphocyte transfer reaction.

BACKGROUND: Initially developed for histocompatibility testing, the normal lymphocyte transfer (NLT) reaction involves the intradermal injection of allogeneic lymphocytes from one individual to another. Because of the unique kinetics of the immunological response to allogeneic lymphocytes, the NLT reaction has been considered an informative system for the analysis of transplant immunity. METHODS: In this study, we used bilateral efferent lymph duct cannulations in sheep to examine the regional lymphatic response to the NLT reaction. Our studies used monoclonal antibodies to define lymphocyte population dynamics and DNA flow cytometry to reflect lymphocyte proliferative responses. RESULTS: The results confirmed a biphasic NLT reaction. An unexpected finding was the marked differences between the early and late NLT responses. The early response was characterized by T-lymphocyte proliferation, as reflected by S-phase DNA, which was comparable in both the NLT-stimulated and contralateral control efferent lymphocytes. This bilateral proliferative response was observed in both CD4+ and CD8+ lymphocytes. In contrast, the late response was restricted to the efferent lymph from the NLT-stimulated lymph node. Dual-parameter flow cytometry demonstrated that the dominant component of this unilateral NLT response was CD8+ lymphocytes. CONCLUSIONS: These results suggest important functional distinctions between systemic and regional lymphatic responses to intradermal alloantigens.

Animals↗

Cold ischaemia time added to kidneys can be minimized by completing the final cross-match before organs are taken from the operating room.

UNLABELLED: Minimizing the amount of cold ischaemia time (CIT) added to cadaveric kidneys before their transplantation is an important goal since longer CIT is associated with worse long-term graft outcome. Our organ procurement organization (OPO) and HLA laboratories have taken the approach of performing the histocompatibility testing, including the final cross-match, as early in the donor process as possible. METHODS: The data in this study were collected from all consecutive final cross-matches done for cadaveric kidney (n = 113) and simultaneous pancreas + kidney (SPK) (n = 25) transplants done with organs recovered from donors in the Midwest Transplant Network OPO from 1 January 2001 to 9 May 2002. We evaluated the time the final cross-match was completed from when the kidneys from that donor were taken from the operating room (OR) and compared that time with CIT. RESULTS: For kidney transplants, 72% of the final cross-matches were complete before the kidneys were taken from the OR. The CIT of that group (10.4 +/- 3.8 h) was significantly lower than that of the group of kidney transplant patients whose final cross-match was done after the kidneys were taken from the OR (15.5 +/- 5.8 h) (P < 0.001). Similarly, for SPK transplants, 88% of the final cross-matches were completed before the organs left the OR and the CIT of that group (10.2 +/- 3.4 h) was less than in the group whose final cross-match was done after the organs left the OR (14.3 +/- 4.8 h) (P > 0.1). CONCLUSIONS: These data show that the practice of completing the final cross-match as early in the donor process as possible helps to minimize the amount of cold ischaemia time added to the kidneys and pancreata before transplantation. That should reduce the detrimental influence that longer CIT has on short- and long-term function in kidney as well as SPK transplantation.

Cold Temperature↗

Immunological reactivity towards chondrocytes in rat and man: relevance to autoimmune arthritis.

Immunological investigation of articular chondrocytes obtained from rat and man have shown the presence of unique differentiation antigens on the cell surface demonstrated by poly- or monoclonal antibodies in both species, and by the analysis of T cell reactivity in the rat. Both species of chondrocytes express Class I antigens in common with all nucleated mammalian cells and, in man, individual specificities of the MHC A, B, and C locus can be identified using standard histocompatibility testing. Class II antigens are strongly expressed in the rat but are expressed poorly in the human specimens we have analyzed. This finding is at variance with the reports of others and may depend upon the antisera we have used or the diseased state of our patient donors. In the rat, T cell reactivity to chondrocyte antigens is strong and can be demonstrated in both proliferation and cytotoxic assay. Moreover, syngeneic reactivity is present in naive animals, suggesting that rats are not tolerant of their own CSDA. The cross-reactivity found on analysis of cytotoxic killing suggests a sharing of differentiation antigens amongst the different strains of rats and possibly a limited polymorphism for this system. In man, conventional assays for T cell reactivity are hampered by the presence of a soluble inhibitor of lymphocyte proliferation released by chondrocytes and as yet unidentified. The poor representation of Class II antigens on our chondrocyte specimens may further contribute to the difficulty in producing proliferation. On the other hand, early success is reported in finding a significant number of T cell clones isolated from the inflammatory membrane of patients with rheumatoid arthritis, which respond to chondrocyte antigens. The possibility that these antigens may play a role in the pathogenesis of inflammatory arthritis may be more readily explored by exploring these approaches.

Animals↗

HLA in allergic twins.

Interest in the study and determination of the influence of a genetic factor in the etiology of bronchial asthma was initiated by Cook. This is a study of 13 pairs of twins that were seen in our department presenting some kind of allergic illness. Each pair of twins underwent a complete clinical history and physical examination, routine laboratory analysis and radiography. In addition, they were also tested for the following: skin test, serum immunoglobulins, total IgE, histamine release test, hemagglutination test, histocompatibility antigen (HLA). Statistical analysis was done using the Chi square test, with significant results found in 4 antigens, namely A2, A30 + 31, B5 and B12.

Adolescent↗

Gregg's congenital rubella patients 60 years later.

BACKGROUND: In 1941, a Sydney ophthalmologist, Norman McAlister Gregg, correctly identified the link between congenital cataracts in infants and maternal rubella early in pregnancy. Fifty of Gregg's subjects with congenital rubella, born in 1939-1944, were reviewed in 1967 and again in 1991. We reviewed this cohort in 2000-2001, 60 years after their intrauterine infection. METHODS: The subjects underwent full clinical assessment, plus pathology tests, an ophthalmological and cardiological review (including electrocardiography and echocardiography) and HLA histocompatibility testing. RESULTS: Since they were first seen in 1967, 10 have died (cardiovascular causes [4], malignant disease [4], AIDS [1], and hepatitis C-related cirrhosis [1]). All surviving men came for review (19) and 13 women (eight women declined). Echocardiography showed mild aortic valve sclerosis in 68%. The prevalence of diabetes (22%), thyroid disorders (19%), early menopause (73%) and osteoporosis (12.5%) was increased compared with the Australian population; 41% had undetectable levels of rubella antibodies. The frequency of HLA-A1 (44%) and HLA-B8 (34%) antigens was increased, and the haplotype HLA-A1, B8, DR3, said to be highly associated with many autoimmune conditions, was present in 25%. CONCLUSIONS: This cohort of people with congenital rubella has illuminated our understanding of viral teratogenesis.

Antibodies, Viral↗

Histocompatible unrelated donors for bone marrow transplantation.

Bone marrow transplantation using HLA one haplotype mismatched family donors has been associated with a high incidence of severe graft-versus-host disease, and an increased risk of graft failure. Recently, the use of histocompatible unrelated donors has been investigated when an HLA genotypically identical sibling is not available. Conventional histocompatibility testing of potential unrelated donors does not always predict alloreactivity in vivo. In this review, the preliminary results of histocompatible unrelated transplants are presented and methods of matching unrelated donors discussed.

Bone Marrow Transplantation↗

[HLA antigens in bipolar affective patients].

The presence of H.L.A. antigens in bipolar affective patients attending in the University Hospital of Zaragoza was studied and contrasted with findings in the general population. A random sample of 36 patients was selected for the study. All of them fulfill, both, DSM-III and RDC diagnostic criteria . The HLA antigen was studied by means of Lymphotoxicity Test of N.I.H. standard technique. Both, A29 and B21 HLA antigens were detect in the bipolar patients more frequently that in the controls (Histocompatibility Testing data, 1980), and the differences were statistically significant. It is also suggested that the A2 HLA antigen is more frequently found in bipolar patients "non-respondent" to lithium treatment. The inclination to relate these antigens to specifics genes in the bipolar illness should be pondered, since the risk of "false positives" inherent in studies testing association hypothesis is well known.

Bipolar Disorder↗

An HLA-D specificity found in the Japanese population.

A new HLA-D specificity was found in the Japanese population in two different laboratories. Japanese cell YT, found at Stanford, California, was A9,BW22J,CW1 and cell Wa, found at Sapporo, Japan, was A9,BW22J homozygous. They were shown to be HLA-D identical to the homozygous Japanese cell AH which submitted to the VIth International Histocompatibility Testing Workshop (Workshop number 2-001). This specificity was common in the Japanese (gf = 0.089) but completely absent from 62 Caucasians tested. Strong association of this specificity with HLA-BW22J was demonstrated.

California↗

Clinical relevance of anti-HLA antibodies pre and post transplant.

Pretransplant histocompatibility testing seeks to select compatible donor-recipient pairs for transplantation. Sera from prospective renal transplant recipients are screened for the presence of human leukocyte antigen (HLA) antibodies to determine humoral alloimmunization. Present techniques screen patient sera using a complement-dependent cytotoxicity assay and express the results as percent of panel reactive antibody (PRA). However, the standard assay suffers because it needs viable target cells, a variable sensitivity of cells for complement, subjective evaluation, a lack of standardized methodology, and a variable correlation with clinical outcomes. Alternatively, an enzyme-linked immunosorbent assay (ELISA) methodology can detect IgG anti-HLA reactivity based on the binding of immunoglobulin to soluble HLA class I antigens. This method provides increased objectivity and reproducibility, does not require use of viable target cells, and most importantly, detects immunoglobulin that is reactive to HLA class I antigens. Data discussed herein suggest that identifying reactive recipient sera using the enzyme-linked immunosorbent assay (ELISA) (PRA-STAT, Sang Stat Med, Menlo Park, CA) methodology may be more informative clinically than current standard percent of panel reactive antibody (PRA) assays.

Cytotoxicity Tests, Immunologic↗

Typing of serum-soluble HLA-B27 antigen by ELISA.

An ELISA using serum as soluble HLA antigen source was developed for HLA-B27 typing. Two sandwich assays were run in parallel. The first assay utilized a monoclonal antibody (mAb) reacting with a determinant expressed by both HLA-B7 and B27 antigens; the other assay utilized a mAb reactive with HLA-B7 antigens but not with HLA-B27 antigens. After incubation with serum samples, bound HLA antigen was detected using an anti-beta 2m antibody conjugated to peroxidase and a chromogenic substrate. Absorbance of each well was measured at 490 nm. Based on analysis of absorbances obtained with panels of specimens of known HLA phenotypes, a mathematical algorithm was developed to derive the specimen HLA-B27 phenotype from its ELISA absorbance values. Despite the lack of monospecific mAb, an accurate HLA-B27 typing was possible. 362 specimens (including 151 HLA-B27-positive) were tested. Agreement between microlymphocytotoxicity and ELISA was 99.2%. No correlation between the level of HLA-B27 antigen reactivity and the amount of total HLA class I antigen in serum was observed. This report demonstrates the possibility of using serum-soluble HLA antigen and ELISA technology for histocompatibility testing. The assay offers several significant advantages over microlymphocytotoxicity: no need for cell preparation, batch testing capabilities and objective, reproducible interpretation of results.

Algorithms↗

Familial rheumatoid arthritis: linkage of HLA to disease susceptibility locus in four families where proband presented with juvenile rheumatoid arthritis.

The occurrence of a chronic seronegative polyarthritis has been studied in four families in which the proband presented with some form of juvenile rheumatoid arthritis. In these families, histocompatibility testing suggested that susceptibility to arthritis was controlled by a dominant allele with variable penetrance and expressivity at the rheumatoid-like arthritis, first locus (RLA-1). The combined lod scores for the four families (2.70) indicated that the odds in favor of genetic linkage between the major histocompatibility complex and the postulated disease susceptibility gene, RLA-1, were 500:1. In one family, a recombinant event permitted localization of RLA-1 centromeric to HLA-D. Of major interest was the fact that there was significant pleomorphism in the clinical manifestations of arthritis in affected individuals. In some, symptoms first occurred in childhood and in others, in adult life. Even among those with childhood-onset arthritis, different types of juvenile rheumatoid arthritis were observed within the same family.

Adolescent↗