Reducing the incidence of TRALI in the UK: the results of screening for donor leucocyte antibodies and the development of national guidelines.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is associated with the passive transfusion of leucocyte antibodies in blood products. Blood Transfusion Services have adopted a number of different strategies for reducing the incidence of TRALI, but, while these have been successful, TRALI has not been completely eliminated. Many Transfusion Services have introduced leucocyte antibody screening of donors to further reduce TRALI. This report describes the results of donor leucocyte antibody screening within NHS Blood and Transplant and the guidelines that have been developed for Transfusion Services within the United Kingdom (UK) to reduce the incidence of TRALI. MATERIALS AND METHODS: Blood samples from newly recruited female apheresis donors were tested for human leucocyte antigens (HLA) class I and class II antibodies and granulocyte-specific antibodies. RESULTS: A total of 1157 female donors were evaluated. Three hundred and fifteen (27·23%) donors had HLA class I or II antibodies and were returned to red cell component donation. Fifty-seven (6·77%) of the remaining 842 donors were found to have granulocyte-specific antibodies of which 11 (1·31%) had HNA-specific antibodies. A total of 818 donors (70·70%) were accepted for platelet apheresis, 336 donors (29·04%) were returned to red cell component donation, and three donors with HNA-3a antibodies (0·26%) were deferred from therapeutic donation. CONCLUSIONS: Female donors with leucocyte antibodies were identified in a stratified screening programme. Donors with antibodies were either directed to red cell donation or deferred. This process, combined with other measures that have already been introduced, is anticipated to further reduce the incidence of TRALI.

Analysis of paternal alleles in nucleated red blood cells enriched from maternal blood.

The purpose of our study was to identify paternal alleles in NRBC enriched from maternal peripheral blood for detection of the presence of foetal cells in the maternal circulation and to establish a reliable non-invasive method which should allow following genetic testing. For enrichment of foetal cells from peripheral maternal blood we combined Ficoll-Paque density gradient centrifugation and MACS. Maternal leukocytes were firstly depleted using anti-CD14 and anti-CD45 microbeads. NRBC were sorted from the CD14-/CD45- fraction by positive selection using CD71 microbeads. Paternal alleles in the CD14-/CD45-/CD71+ fraction were indicated by the PCR method using HLA (DRB1, DQB1, DQA1) and Polymarker System (LDLR, GYPA, HBGG, D7S8, GC) as genetic markers. Different paternal alleles of studied 8 loci were detected in 13 out of 19 samples of cells enriched from maternal peripheral blood between the 13th and 36th week of gestation. Our results demonstrate that foetal cells enriched from maternal peripheral blood may be used as a source of foetal DNA for prenatal diagnosis, paternity testing and other application.

Identification of HLA alleles with low or no cell surface expression in the Czech population.

The presence of the A*24020102L allele is implicated in one donor from the CBMD who serologically was typed as A2; B44, B55; Cwl, Cw7. The DRB4*01030102N allele was identified in one healthy donor and in one patient with MDS during routine HLA class II DNA typing. The DRB4*01030102N allele was identified in the patient's father, who had CML, and was associated with the HLA-A3-B7-Cw7-DRB1*0701-DQB1*0303 haplotype, which is common for European populations. In order to avoid mistyping, both techniques, serology and molecular biology must be used for HLA typing, especially for cases where just one antigen appeared to be present using serological methods.

The Czech Bone Marrow Donor Registry.

OBJECTIVE: The Czech Bone Marrow Donor Registry (CBMD)--established 9 years ago, operates within the National HLA Centre, a constituent part of the Department of Immunology at the Institute for Clinical and Experimental Medicine. The Czech Cord Blood Register (CSCB) was recently established (in 1996) and started its activities. METHODS: CBMD is responsible for maintaining a database of HLA typed volunteer donors, for performing national and international searches in the file of bone marrow transplantation as well as for coordinating the communication between participating centres. RESULTS: The operation of the CBMD registry requires the modern communication technology for the exchange of data with local organisations (donor and transplant centres) and with international BM organisations and networks abroad (Bone Marrow Donors Worldwide--BMDW, National Marrow Donor Program--NMDP, European Donor Secretariat E.D.S., European Marrow Donor Information System--EMDIS). CONCLUSIONS: The CBMD is fully integrated into international cooperation. The HLA typed unrelated stem cells from Prague can be selected for patients in the whole world.

[Characterization of null alleles in human histocompatibility complex antigens (HLA)]. / Charakterizace nulových alel v komplexu lidských histokompatibilních antigenu (HLA).

The widespread application of DNA techniques in medicine and biology has allowed the typing of human leukocyte antigens (HLA) at the molecular level. Comparative studies between serological and molecular biology methods have shown the existence of null alleles, which code HLA antigens with low or no cell surface expression. Null alleles are not detectable by standard serological typing methods and may be overlooked/or incorrectly assigned by available DNA methods. Although null alleles are infrequent in human populations, they should not be ignored. Errors in typing of null alleles may cause complications in the evaluation of HLA matching of donor/recipient pairs that were originally considered HLA-compatible. The detection of null alleles and their frequency in various populations requires typing of a large number of individuals by both serological and DNA-based methods. Knowledge of the haplotype(s) associated with null alleles may be helpful for their identification. For this purpose, it is necessary to perform family studies.

[Prediction of insulin-dependent diabetes mellitus in children of first-degree relatives of diabetic patients]. / Predikce inzulin dependentního diabetes mellitus u detských prvostupnových príbuzných diabetických pacientu.

BACKGROUND: Individuals at risk for insulin dependent diabetes mellitus (IDDM) can be identified using a combination of genetic, immunological and metabolic markers. Our study was aimed at prediction of IDDM in a cohort of children having a first-degree relative with IDDM. METHODS AND RESULTS: In the period of three years, we investigated 208 non-diabetic children and adolescents, aged 10.0 +/- 5.3 (mean +/- SD), mostly siblings of diabetic children. The genetic risk was determined by the HLA-DQB1, -DQA1 genotyping and subtyping of the DRB1*04 alleles carried on the DQB1*0302 haplotypes. Insulitis was detected using a combination of autoantibody tests against three molecular-defined antigens (insulin, GAD65, IA-2). Prevalence of insulitis (defined as confirmed positivity of at least one autoantibody) was 9/208 (4.3%). In children carrying the IDDM highest-risk genotype (HLA-DQB1*0201-DQA1*05/DQB1*0302-DQA1*03), insulitis was almost 10 times more frequent (5/24, 21%) than in children with other genotypes (4/184, 2.2%, P = 0.003). In all subjects with insulitis, the first phase insulin response (FPIR) was determined by the intravenous glucose tolerance test. Three of the nine children had decreased FPIR, of whom two were later diagnosed with IDDM. None of the remaining children developed IDDM. CONCLUSIONS: We present the first IDDM prediction study in the Czech population, emphasising the utility of genetic risk investigation in the prediction scheme.

A chromosomal event resulting in possession and expression of multiple HLA-B and Cw genes in a renal patient.

Routine HLA typing of a renal patient for purposes of registration for transplantation revealed an unusual human leucocyte antigen (HLA)-B and Cw genotype, with three specificities detected. Results were confirmed in a second sample, and in a second laboratory. The possibility of these results reflecting a chimaeric state was rejected following short tandem repeat (STR) analysis. Although cytogenetic analysis has failed to detect a chromosomal abnormality, these findings support the view that the aberrant expression of HLA in this patient resulted from an unequal crossover event, occurring during meiosis in a previous generation.

Identification of a novel HLA-A allele (A*1115) in the UK National External Quality Assessment Schemes for Histocompatibility and Immunogenetics' Educational Cell Exchange.

The novel allele, HLA-A*1115, was identified in an 'Educational Scheme' sample (ED03/03 - from a north-western European Caucasoid blood donor) distributed by the UK National External Quality Assessment Schemes for Histocompatibility and Immunogenetics. ED03/03 was typed by serology, the polymerase chain reaction using sequence-specific primers and sequence-based typing. A*1115 is most similar to A*110101 with a single mismatch (G to C) at constant position 565, leading to a conservative amino acid change from valine (GTG) to leucine (CTG) at codon 165 in the alpha(2) domain. This substitution has not been reported for any other HLA class I allele so far. The HLA-A*1115-bearing haplotype was B*350101; Cw*040101; DRB1*140101; DRB3*020201; DQA1*010401; DQB1*0503; DPA1*0103/07; DPB1*030101. Extensive serological typing indicated that this allele essentially encodes a 'normal' HLA-A11 specificity.

HLA-DQB1 sequencing-based typing using newly identified conserved nucleotide sequences in introns 1 and 2.

Sequencing-based typing (SBT) human leukocyte antigen (HLA) class I and II genes should examine entire exon sequences where polymorphisms lie. Primers for the amplification of complete exons therefore anneal in introns and their design relies on accurate intron sequences being available. We decided to develop a SBT method for HLA-DQB1 using amplification primers which anneal in introns 1 and 2, yet the amount of intron sequence data previously available in databases was sparse. Therefore, we undertook a systematic sequencing of introns 1 and 2 using DNA from cell lines homozygous for DQB1. This study confirmed an earlier report that the non-coding regions of this gene are the most polymorphic seen in the human genome. Intron sequences within an allele group were largely identical, the exceptions being DQB1*0301 differing from other DQB1*03 allele groups and DQB1*0601 differing from all other DQB1*06 alleles. A retroviral Alu element, related to the AluYa5a2 subfamily, was identified uniquely inserted in intron 2 of DQB1*02 alleles. For the typing approach, six amplification primers were designed based on conserved allele group sequences covering all of the HLA DQB antigens, and two sequencing primers were also designed which anneal in intron 2. This method has proved to be very robust and has been used as part of a referral DNA sequencing service for a number of years.

A new HLA-A allele, HLA-A*6824, identified in three unrelated individuals.

A novel allele, human leukocyte antigen (HLA)-A*6824, has been identified in three unrelated individuals of northwestern European origin in a period of less than 4 months, implying that this allele may be quite common in this population. HLA-A*6824 differs from A*680102 by a single nucleotide change at position 275 in exon 2, which results in a conservative amino acid substitution from lysine to arginine in the peptide-binding groove at codon 68.

Georgian and kurd mtDNA sequence analysis shows a lack of correlation between languages and female genetic lineages.

Mitochondrial DNA sequences from Georgians and Kurds were analyzed in order to test the possible correlation between female lineages and languages in these two neighboring West Eurasian groups. Mitochondrial sequence pools in both populations are very similar despite their different linguistic and prehistoric backgrounds. Both populations present mtDNA lineages that clearly belong to the European gene pool, as shown by 1) similar nucleotide and sequence diversities; 2) a large number of sequences shared with the rest of European samples; 3) nonsignificant genetic distances; and 4) classification of the present lineages into the major European mtDNA haplogroups already described. The outlier position of the populations from the Caucasus according to classical genetic markers is not recognized in the present Georgian mtDNA sequence pool. This result suggests that the differentiation of mtDNA sequences in West Eurasia and the outlier features of Caucasian populations should be attributed to different processes. Moreover, the putative linguistic relationship between Caucasian groups and the Basques, another outlier population within Europe for classical genetic markers, is not detected by the analysis of mtDNA sequences.

[Circulating immune complexes and neutrophil function in low-birth-weight newborn infants in the early neonatal period]. / Tsirkuliruiushchie immunnye kompleksy i funktsional'naia aktivnost' neitrofilov u novorozhdennykh s nizkoi massoi tela v rannem neonatal'nom periode.

Peripheral blood neutrophil function and the content of circulating immune complexes in funic blood were examined in 35 premature neonates with low body weight, 27 neonates with grade I prematurity and in 24 normal full-term neonates (a control group) by days 1-2 and days 7-8 of life. The positive time-course of changes in the neutrophil capacity for completed phagocytosis and in the content of circulating immune complexes was disclosed in full-term neonates with low body weight at birth. The same parameters showed a negative time-course of changes in premature neonates.

[Immunologic indicators in mothers of low birth weight infants]. / Immunologicheskie pokazateli materei novorozhdennykh s nizkoi massoi tela pri rozhdenii.

Neutrophil function, circulating immune complexes, major immunoglobulins and lysozyme activity have been determined in mothers of 86 newborns, of whom 24 were term infants with normal birthweight, 27 had first-degree prematurity and 35 were term newborns with low-birth weight. The mothers of premature newborns showed prominent impairment of bactericidal properties and functional stores of neutrophilic phagocytes, while mothers of term newborns with low-birth weight had abnormalities of humoral immunity.