International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012).

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

Human platelet-specific antigens frequencies in the Argentinean population.

The frequencies of several human platelet antigens (HPAs) vary between different populations and are a major determinant for the prevalence of HPA alloimmunization and its clinical associated entities. The aim of this study was to characterize the allele frequencies of seven HPA systems in two different ethnic groups from the Argentinean city of Rosario, the major population and a minority Amerindian group recently arrived from the north of the country, the Tobas. A total of 192 healthy unrelated individuals from blood donors and hospital staff from the Hospital Italiano Garibaldi and 27 unrelated Toba Amerindians were genotyped for HPA-1, -2, -3, -4, -5, -6 and -15 systems by polymerase chain reaction with sequence specific primers (PCR-SSP). The present data showed that the distribution of the HPA alleles among Argentineans from Rosario is quite similar to that reported among Europeans. The frequencies seen in Tobas, although limited by the small number of aboriginal samples studied, are similar to those reported for other Amerindians populations. Statistically significant differences were found for the genotype distribution of HPA-1, -3, -5 and -15 between both groups, indicating important differences in the potential risk of HPA alloimmunization associated to transfusion and pregnancy. The study of these polymorphisms represents the first step in the elucidation of pathological conditions that are underdiagnosed in our population. It allowed us to establish a panel of characterized blood donors necessary for the serological work out and as a source for compatible platelets transfusion.

International society of blood transfusion working party on red cell immunogenetics and terminology: report of the Seoul and London meetings.

The Working Party has met twice since the last report: in Seoul, South Korea 2014, and in London, UK 2015, both in association with the International Society of Blood Transfusion (ISBT) Congress. As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. Eleven new blood group antigens were added to seven blood group systems. This brings the current total of blood group antigens recognized by the ISBT to 346, of which 308 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known blood group system.

Placenta-specific expression of the rat growth hormone-releasing hormone gene promoter in transgenic mice.

GH-releasing hormone (GHRH) is a hypothalamic peptide that plays a critical role in controlling the synthesis and secretion of GH in the anterior pituitary. Along with many other hypothalamic hormones, GHRH is also expressed in the placenta, although its physiological role in this tissue has not yet been determined. The placental prepro-GHRH is identical to that found in the hypothalamus. However, the placental and hypothalamic GHRH messenger RNAs differ in the region corresponding to the untranslated exon 1. A combined mechanism involving the use of tissue-specific promoters and the differential splicing of exon 1 generates the mature GHRH messenger RNAs in placenta and hypothalamus. As a first step toward the localization of the regulatory elements involved in the placenta-specific expression of the GHRH gene, we have generated transgenic mice containing constructs in which potential regulatory sequences of the rat GHRH gene were fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Construct GHRH-CAT1, which contains 7.5 kilobases of flanking sequences upstream to the placental transcription start site, did not promote CAT expression in the transgenic animals. In contrast, construct GHRH-CAT2, which differs from construct GHRH-CAT1 in having additional sequences located downstream to placental exon 1, exhibited high levels of CAT expression in brain and placenta. Our results show that the sequences included in construct GHRH-CAT2 contain the cis-acting regulatory elements necessary to direct developmentally regulated and cell type-specific expression of the CAT gene in the placenta. Unexpectedly, the expression of the transgene in the brain was detected in glial cells of different areas, but not in the hypothalamus.

Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli.

Two fragments of a cDNA encoding radish 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET-8c and expressed in Escherichia coli. The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity. In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form. The amount of soluble active enzyme in cell-free extracts of E. coli dramatically increased when the temperature during the induction was lowered from 37 degrees C to 22 degrees C.

Expression of a fusion gene consisting of the mouse growth hormone-releasing hormone gene promoter linked to the SV40 T-antigen gene in transgenic mice.

Limited information is available concerning the regulation of growth hormone-releasing hormone (GHRH) gene expression in the hypothalamus, largely because of the lack of a suitable cellular model. In an attempt to immortalize hypothalamic GHRH-producing neurons, we have generated a transgenic mouse model which expresses the simian virus 40 (SV40) T-antigen gene (Tag) under the control of the GHRH gene promoter. The transgene contains approximately 5 kb of mouse GHRH gene sequences, including 3.5 kb of the 5'-flanking region, the entire hypothalamic exon 1 and 1.5 kb of intron 1, fused to the SV40 Tag gene. This construct was microinjected into fertilized oocytes. Fourteen of 96 mice born had integrated the transgene. These mice were fertile and showed no signs of central or peripheral tumors. The pattern of expression of the SV40 Tag gene was analyzed in four different transgenic lines by RT-PCR. The tissues tested include: hypothalamus, pituitary, cortex, cerebellum, spinal cord, adrenal, testis, spleen and lung. Transgene expression was consistently detected in the hypothalamus of all lines. In addition, SV40 Tag expression was also detected in the hypothalamus by Northern blot analysis in two of the transgenic lines. SV40 Tag expression was also detected in the testis of all transgenic lines by RT-PCR. This result was not expected since the GHRH gene sequences present in the transgene do not include the testis-specific transcription initiation site previously described. This suggests that GHRH gene expression in the mouse testis can be directed by regulatory sequences located downstream of the testis specific transcription start site. We conclude that the promoter region of the GHRH gene included in this construct contains the regulatory elements necessary to drive hypothalamic and testis expression in vivo. In addition, all mice from one of the transgenic lines developed cataracts in both eyes. SV40 Tag expression was detected not only in eyes with cataracts, but also, to a lesser extent, in eyes from other transgenic lines. Furthermore, the endogenous GHRH gene was found to be expressed in the eyes of normal mice.

HNA-1a, HNA-1b and HNA-1c gene frequencies in Argentineans.

Human neutrophil antigens (HNA) are polymorphic structures located in the neutrophil membrane. The neutrophil-specific antigens HNA-1a (NA1), 1b (NA2) and 1c (SH) are well-recognized allotypic forms of FcgammaRIIIb and the most frequent targets of neutrophil alloantibodies. The aim of this study was to determine the gene frequencies of the neutrophil-specific antigens belonging to the HNA-1 system in blood donors and Toba Amerindians from Rosario, Argentina. Two hundred and eighteen unrelated healthy Argentinean blood donors and Toba Amerindians from Rosario were typed for HNA-1a, HNA-1b and HNA-1c using polymerase chain reaction with sequence-specific primers. For the Argentinean blood donors, the HNA-1a and HNA-1b gene frequencies were 0.44 and 0.56 and for the Amerindians Toba were 0.77 and 0.23, respectively. The HNA-1c antigen is present in 4.7% (gene frequency=0.023) of the blood donors but in none of the Amerindian individuals. The present data showed that the HNA-1 allele frequencies in the major population and the Toba Amerindians from Rosario are similar to those described in European and others distant Amerindians populations, respectively.

Comparison of three microtube column agglutination systems for antibody screening: DG Gel, DiaMed-ID and Ortho BioVue.

The aims of the present study were to evaluate the estimated diagnostic accuracy of a new microtube column agglutination system (DG Gel, Diagnostic Grifols, Barcelona, Spain), to analyse the antibody reactivity and to compare the data with the two well-established DiaMed-ID and Ortho BioVue systems. We collected 3024 consecutive samples from blood donors, transfusion recipients and pregnant women, and 100 samples containing antibodies of known specificity. All these samples were tested in parallel by the three microtube agglutination systems. The estimated sensitivity was 100% for DG Gel and Ortho BioVue and 97.58% for DiaMed-ID. The estimated specificity was 99.93% for Ortho BioVue and 100% for DiaMed-ID and DG Gel. The score mean and range of the antibody titration of DG Gel, DiaMed-ID and Ortho BioVue were 34.31 (5-119), 30.3 (3-121) and 37.38 (3-112), respectively. All three column agglutination systems work well showing a high estimated diagnostic accuracy.

Identification of a new HLA-DRB4 allele (DRB4*01033) by PCR-SSP and direct sequencing.

In this report, we describe the identification of a novel DRB4*01 allele, DRB4*01033, found in two Spanish Caucasian individuals. The new allele was detected during routine HLA typing by an unusual pattern of amplification obtained by polymerase chain reaction using sequence-specific primers (PCR-SSP) that did not match with any of the previously described DRB4 alleles. In order to establish the polymorphism responsible for this pattern exons 2 and 3 of the DRB4 locus were amplified and directly sequenced. The new DRB4*01 allele is identical to DRB4*0103101 except for a single nucleotide substitution in codon 78 (TAC-->TAT). This nucleotide change does not cause an amino acid change as both triplets code for a tyrosine.

Identification and sequence characterization of a novel HLA-A11 serological variant (HLA-A*1108).

In this report we describe the identification of a novel HLA-A*11 allele, HLA-A*1108, found in two individuals of a Spanish family. This new allele was detected during routine HLA typing by an atypical serological reactivity pattern and by inconclusive patterns obtained in DNA-based typing methods. The nucleotide sequence of exons 2 and 3 of HLA-A*1108 was identical to HLA-A*11011 except for two nucleotide substitutions at codons 152 (GCG-->GAG) and 156 (CAG-->CGG). These mutations change the non-charged amino acid alanine, at codon 152, to negative glutamic acid, and also non-charged glutamine, at codon 156, to positive arginine, which may explain its anomalous serological reactivity.

Identification of a new HLA-DRB1*04 allele, DRB1*0437.

In this report we describe the identification of a novel DRB1*04 allele, DRB1*0437, found in a Spanish individual. The routine HLA typing, in the context of bone marrow transplantation, by polymerase chain reaction-sequence-based typing (PCR-SBT) made possible the identification of this new allele. This allele is identical to DRB1*0402 except for a single nucleotide substitution at position 286 (A-->C), changing the encoded Isoleucine to a Leucine. The DRB1*0437 allele conserves the same two acidic residues at codons 70 and 71 that confer to DRB1*0402 its association to some autoimmune diseases.

Identification of a novel HLA-DQB1*06 allele, DQB1*0618.

We report here the identification of a novel DQB1*06 allele, DQB1*0618, found in a bone marrow donor. The new allele was detected during routine DNA-based HLA typing by an ambiguous pattern of probe hybridization, obtained by polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO). Molecular cloning and sequencing confirmed that the new allele is identical to DQB1*0609 at exon 2 except for 3 nucleotide substitutions at positions 353, 356 and 367, also found in other alleles. These nucleotide changes may explain its anomalous reactivity.