Detection of granulocyte-reactive antibodies: a comparison of different methods.

BACKGROUND AND OBJECTIVES: Granulocyte-reactive antibodies can cause autoimmune and neonatal immune neutropenias as well as transfusion-related acute lung injury. The classical antibody-detection methods granulocyte aggregation test (GAT), granulocyte immunofluorescence test (GIFT) and monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are time-consuming and technically challenging. In recent years, flow cytometric white blood cell immunofluorescence test (Flow-WIFT) and the microbeads assay LabScreen® Multi have emerged and are still subject of evaluation. These serological tests were compared on a screening and specification level. MATERIALS AND METHODS: For screening, the combination of GAT/GIFT was compared to Flow-WIFT testing 333 samples. Positive samples were further analysed with MAIGA and LabScreen® Multi. RESULTS: Granulocyte aggregation test/GIFT detected 77 positive samples, Flow-WIFT found 108 granulocyte-reactive samples. Six Samples were only positive in GAT/GIFT, and 37 samples were only positive in Flow-WIFT (κ = 0.682). Antibody specification with MAIGA and the microbeads assay confirmed granulocyte-reactivity in 83 cases with 70 matching results (κ = 0.742). However, out of six detected human neutrophil antigen (HNA) reactivities only two specificities matched in both assays. CONCLUSION: Flow-WIFT may be a valuable addition to GIFT for granulocyte-reactive antibody screening. MAIGA remains the most reliable laboratory method for antibody specification.

Granulocyte-reactive antibodies are associated with red blood cell alloimmunization.

Granulocyte-reactive antibodies may cause transfusion-related acute lung injury (TRALI) and immune neutropenias. Risk factors for their acquisition other than previous alloexposition are largely unknown. In addition to the known association between human leucocyte antigen alloantibodies and red blood cell alloimmunization in selected cohorts of transfused patients, this study investigated a possible extension of this association to granulocyte-reactive antibodies in women with a history of pregnancy. The overall prevalence of granulocyte-reactive antibodies in 333 samples from women with a history of pregnancy (143 samples containing red cell alloantibodies) was 23·1%. The prevalence in the red cell-alloimmunized group (32·9%) was significantly higher than in controls (15·8%, P < 0·001). This could suggest that some individuals may be strong immunological responders, forming alloantibodies more readily than others.

High-throughput multiplex PCR genotyping for 35 red blood cell antigens in blood donors.

BACKGROUND AND OBJECTIVES: One to two per cent of patients in need of red cell transfusion carry irregular antibodies to red blood cell (RBC) antigens and have to be supplied with specially selected blood units. To be able to respond to those requests, blood centres have to screen a significant number of donors for a variety of antigens serologically, which is a costly and through the shortage of reagents, also limited procedure. To make this procedure more efficient, the Austrian Red Cross has developed a genotyping assay as an alternative approach for high throughput RBC typing. MATERIALS AND METHODS: A multiplex polymerase chain reaction (PCR) assay was designed for typing 35 RBC antigens in six reaction mixes. The assay includes both common as well as high-frequency-alleles: MNS1, MNS2, MNS3 and MNS4; LU1, LU2, LU8 and LU14; KEL1, KEL2, KEL3, KEL4, KEL6, KEL7, KEL11, KEL17 and KEL21; FY1, FY2, FYB(WK) and FY0 (FYB(ES)); JK1 and JK2; DI1, DI2, DI3 and DI4; YT1 and YT2; DO1 and DO2; CO1 and CO2; IN1 and IN2. The assay was validated using 370 selected serologically typed samples. Subsequently 6000 individuals were screened to identify high frequency antigen (HFA)-negative donors and to facilitate the search for compatible blood for alloimmunized patients. RESULTS: All controls showed complete concordance for the tested markers. The screening of 6000 donors revealed 57 new HFA-negative donors and the blood group database was extended by approximately 210,000 results. CONCLUSION: The study shows that in practice, this high-throughput genotyping assay is feasible, fast and provides reliable results. Compared to serological testing, this molecular approach is also very cost-efficient.

Plasmodium falciparum malaria and the immunogenetics of ABO, HLA, and CD36 (platelet glycoprotein IV).

Plasmodium falciparum malaria has long been a killer of the young, and has selected for polymorphisms affecting not only erythrocytes, but the immunogenetics of three histocompatibility systems: ABO, human leukocyte antigen (HLA), and CD36. The ABO system is important because the original allele, encoding glycosylation with the A sugar, acts as an adhesion ligand with infected red blood cells (iRBC), thereby promoting vasoocclusion. The prevalence of blood group O, which reduces this cytoadhesion, has increased in endemic areas. Other adaptations which could mitigate A-mediated rosetting include weaker A expression and increased soluble A secretion. The role of the HLA system in malaria has been harder to verify. Although HLA-B53 and DRB1*04 may be associated with clinical outcome, HLA studies are challenged by numerous comparisons in this most polymorphic of systems, and confounded by increasingly heterogeneous populations. Certain HLA markers may also reflect linkage artefact with other malaria-relevant polymorphisms. HLA may be less important because the parasite predominantly invades a compartment which does not express HLA. Adhesion of iRBCs is also mediated by CD36, expressed on platelets, monocytes, and microvascular endothelium. CD36 on monocytes is involved in clearing iRBC, while CD36 on platelets and the endothelium may play a role in tissue sequestration. The genetics of CD36 expression are complex, and recent research is fraught with inconsistent results. The solution may lie in examining genotype-phenotype correlations, zygosity effects on differential tissue expression, or other mechanisms altering CD36 tissue expression. Carefully designed prospective studies should bridge the gap between in-vitro observations and clinical outcomes.

A novel HLA-C allele, Cw*0617.

Sequencing analysis of exons 1-3 of the human leukocyte antigen (HLA)-C gene showed a novel allele, HLA-Cw*0617. While the amino acid sequence is identical with the HLA-Cw*060201 allele, the leader peptide differs in three amino acids.

Graft versus host disease after orthotopic liver transplantation documented by analysis of short tandem repeat polymorphisms.

We report a case of graft versus host disease after liver transplantation in which the diagnosis was made by short tandem repeat analysis. A retrospective analysis using a bone marrow sample showed the presence of chimerism already at a time when the characteristic full clinical picture of graft versus host disease had not yet developed, opening the way for the early diagnosis and treatment.

DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis.

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. A previous recommendation published in 2001 has already addressed Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). Since then, the use of Y-STRs has become very popular, and a numerous new loci have been introduced. The current recommendations address important aspects to clarify problems regarding the nomenclature, the definition of loci and alleles, population genetics and reporting methods.

DNA commission of the International Society of Forensic Genetics: Recommendations on the interpretation of mixtures.

The DNA commission of the International Society of Forensic Genetics (ISFG) was convened at the 21st congress of the International Society for Forensic Genetics held between 13 and 17 September in the Azores, Portugal. The purpose of the group was to agree on guidelines to encourage best practice that can be universally applied to assist with mixture interpretation. In addition the commission was tasked to provide guidance on low copy number (LCN) reporting. Our discussions have highlighted a significant need for continuing education and research into this area. We have attempted to present a consensus from experts but to be practical we do not claim to have conveyed a clear vision in every respect in this difficult subject. For this reason, we propose to allow a period of time for feedback and reflection by the scientific community. Then the DNA commission will meet again to consider further recommendations.

Impact of HLA class I high-resolution mismatches on chronic graft-versus-host disease and survival of patients given hematopoietic stem cell grafts from unrelated donors.

There is consensus that matching of unrelated donors (URD) and patients for HLA class II alleles improves the outcome of hematopoietic stem cell transplantation (HSCT). However, the significance of HLA class I allelic mismatches for transplant outcome is under discussion and reports on long-term effects like chronic graft-versus-host disease (GVHD) are rare. Thus, we investigated the association of human leukocyte antigen (HLA) class I allele mismatches and outcome in 144 patients given HSCT from URD who were matched for HLA-DRB1, DRB3/4/5, and DQB1 alleles. The risk of chronic GVHD was significantly increased in patients with class I mismatched donors, the mismatch either detected by low- or high-resolution typing. A single HLA class I allele mismatch significantly increased the risk of chronic GVHD in multivariate analysis. Overall survival was significantly reduced in patient/donor pairs with more than one-allele class I mismatch. Thus, selection of unrelated donors for transplantation should be based on high-resolution HLA class I typing.

ACTBP2 (alias ACTBP8) is localized on chromosome 6 (band 6q14).

The locus ACTBP2 (SE33) is localized on chromosome 6 (band 6q14). This has been demonstrated by typing a large Caucasoid three-generation kindred of Austrian origin for SE33 and several chromosome 6 markers.

Blood transfusion in Europe--The White Book 2005: the patchwork of transfusion medicine in Europe.

In this article, some points of the patchwork of Transfusion Medicine in Europe will be briefly discussed: blood organizations, donor selection and blood donation, training and education, hemovigilance.

The reality of self-sufficiency.

In this article, some points concerning the self-sufficiency in Europe will be discussed. After the definition of the self-sufficiency, the situation in Central and Eastern Europe will be briefly presented, as well as the problems connected with national and community self-sufficiency.

Immunogenicity of human insulin (Novo) or pork monocomponent insulin in HLA-DR-typed insulin-dependent diabetic individuals.

The immunogenicity of human insulin (Novo, Monotard, Actrapid) or pork monocomponent (MC) insulin (Monotard, Actrapid) was studied in 102 HLA-DR-typed patients with newly diagnosed insulin-dependent diabetes mellitus (type I diabetes). After 6 mo of treatment, IgG-insulin antibodies were found in only 14% of the patients receiving homologous MC insulins, but in 29% of the patients on heterologous MC insulins. IgG-insulin antibody titers were significantly lower in patients treated with human insulin compared with diabetic patients who received the corresponding pork MC insulin preparations from the onset of their disease. The previously reported strong influence of immunogenetic factors in determining the magnitude of the anti-insulin immune response was supported by the findings obtained in the pork MC insulin-treated diabetic individuals. Incidence of circulating immune complexes was 14% and 5% after 3 and 6 mo, respectively, of treatment with human insulin, which is considerably lower than previously reported in patients using heterologous non-monocomponent insulin preparations.

West Nile virus lineage 2 infection in a blood donor from Vienna, Austria, August 2014.

Eastern Austria is neighbouring regions with ongoing West Nile virus (WNV) transmissions. Three human WNV infections had been diagnosed during the past decade in Austria. The Austrian Red Cross Blood Service (ARC-BS) started a first voluntary screening for WNV in blood donors from Eastern Austria by Nucleic Acid Testing (NAT) in June 2014. This is also the most extensive WNV surveillance programme in humans in Austria so far. In August 2014, one autochthonous WNV infection was detected in a blood donor from Vienna. By now, one in 67,800 whole blood donations was found to be positive for WNV RNA.

Paternity testing--quo vadis?

The basis and efficiency of paternity testing using conventional markers (alloantigens or electrophoretically defined polymorphisms in blood) for individuals involved in cases of affiliation are shown. A possible use of DNA polymorphisms in paternity cases is discussed. Due to the fact that the formal genetics of DNA polymorphisms have not been fully validated by the analysis of large numbers of informative meioses, it is not yet possible to include these systems in routine paternity testing. As the vast majority of cases can be resolved by conventional markers, the inclusion of DNA polymorphisms is not usually necessary; in some cases, however, they can provide additional information.

Stimulation of spermatogenesis and biological paternity by intranasal (low dose) gonadotropin-releasing hormone (GnRH) in a male with Kallmann's syndrome: intraindividual comparison of GnRH and gonadotropins for stimulation of spermatogenesis.

Intranasal (in) GnRH spray caused induction and maintenance of spermatogenesis and biological paternity in a 28-yr-old man with Kallmann's syndrome. Prior treatment had included GnRH analog administration, which failed to induce puberty, and testosterone (T) enanthate weekly. Prior hCG/human menopausal gonadotropin therapy had resulted in high normal serum T levels and near-normal semen quality, but during subsequent hCG therapy, spermatogenesis markedly decreased. The patient had then received 250 mg T enanthate/month for 2 yr and 7 months; it was discontinued 7 weeks before the in GnRH study began. At its start (July 1984) the subject's testis size was 7 mL, and he had azoospermia, low serum LH and FSH levels, and a serum T of 74 ng/dL (2.6 nmol/L). GnRH was administered in a dose of 200 micrograms (20-120 ng/kg were absorbed into the circulation) every 2 h, seven to nine times a day, between 0700 and 2400 h for 242 days. After 12 days of treatment, serum T had increased to 519 ng/dL (18.0 nmol/L). After 70 days, the patient's sperm count was 11.5 million/mL (4.5 mL ejaculate volume; 70% motility; 36% normal morphology); on day 185, sperm count was 31 million/mL (4 mL ejaculate volume; 54% motility; 36% normal morphology). His spouse conceived on day 162 and delivered a fullterm daughter 265 days later. The probability of paternity was 99.9994%. Our results suggest that induction and maintenance of spermatogenesis as well as fertility in hypothalamic hypogonadism can be achieved with in GnRH therapy if pituitary and testicular function are intact. Spermatogenesis induced by in GnRH has the same quality as spermatogenesis induced by hCG/human menopausal gonadotropin therapy. Patient compliance is probably the most important factor for the success of in GnRH therapy.