The study was purposed to analyze DNA and allele structure of the partial D phenotypes D(Va) and D(VI) of the Rhesus blood group in Chinese. Through polymerase chain reaction (PCR) and direct genomic DNA sequencing, the RHD gene was detected in three weak D individuals identified serologically. The results showed that among the three weak D individuals, one was identified as partial D phenotype D(Va) (Hus) type and genotyped DccEe; another two were testified as D(VI) III type and genotyped DCcee. Moreover, the breakpoints of the replaced region by RHCE in D(Va) (Hus) were 5' end of the exon 5 and 3' end of the intron 5, and there were 7 novel polymorphisms in intron 5: 23-25(GCA)2, 98G>A, 168-169insG, 205-206insT, 494-495insA, 1256-1257insC, 1347G>T. In conclusion the whole exon 5 and intron 5 are replaced by RHCE in D(Va) (Hus) detected in Chinese.
China ; Exons ; genetics ; Genotype ; Humans ; Introns ; genetics ; Phenotype ; Polymerase Chain Reaction ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA

There are 3' non-coding region, downstream Rhesus box, SMP1 gene et cetera. after RHD stop code. This study was intended to determine the sequence of 3' non-coding region. One pairs of primer was designed and then a polymerase chain reaction (PCR) was established for specific amplification of whole length of 3' non-coding region of RHD in 10 Rh-positive and 10 D(el) samples. The PCR products were purified and directly sequenced. The results showed that all Rh-positive and D(el) samples were identical, which revealed that there were 103 bp between 3'-end of RHD coding region and 5'-end of downstream Rhesus box. The D(el) samples showed the same result with the normal Rh-positive sample. It suggests that lower expression of D antigen in D(el) red cells does not associate with 3' non-coding region of D(el) gene.
3' Untranslated Regions ; genetics ; Base Sequence ; Humans ; Molecular Sequence Data ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA

OBJECTIVEPreviously the weak D and D category allele were investigated in Caucasian and Japanese families. The current study is aimed at an RHD positive, D antigen negative allele in a Chinese family.

METHODSA pair of primers specific for RHD 270A allele were designed, and a sequence specific primer-PCR (SSP-PCR) method was then established to detect RHD 270A allele in 6 members of a family. Furthermore, RFLP method was used to determine the RHD zygosity in all family members.

RESULTSThe RHD 270A allele was detected in the proband, her father and uncle but not grandmother. Therefore this allele may be from grandfather and is inherited through 3 generations. The RHD zygosity test showed that the father and uncle possess one normal RHD gene as RHD 270A carriers, the mother is RHD(+)/RHD(-)heterozygote and the individual is RHD 270A/RHD(-)which causes an RHD positive, D antigen negative trait.

CONCLUSIONThe RHD 270A allele is an ancestral allele, but not a spontaneous.

Alleles ; Asian Continental Ancestry Group ; genetics ; Female ; Genotype ; Humans ; Male ; Pedigree ; Rh-Hr Blood-Group System ; genetics

Flow cytometry was previously applied for analysis of Rh(D) antigen density, therefore it was suggested that the flow cytometry may be used for routine detection of weak D positive phenotypes. This study was purposed to evaluate its practicability. Six weak D positive and 7 DEL individuals were detected by using saline, IAT and absorption/elution test from 2010 to 2011 years. By RHD genotyping, zygosity analysis and sequencing, 3 cases of weak D type 15, 3 cases of partial D type DVI-III and 7 cases of DEL carrying RHD1227A alleles were identified. Taking 2 normal Rh(D)-positive and 2 D-negative samples as controls, all the samples were tested by using flow cytometry, and the median fluorescence intensities were observed as well. The results indicated that all weak D type 15 and partial D type DVI samples were detected to be positive by flow cytometry, as compared with 2 Rh(D)-negative samples (P < 0.05). Seven 7 DEL samples were tested to be negative (P > 0.05), although one of 7 DEL was tested as "±" in IAT and strong positive in absorption/elution. The RHD zygosity analysis showed this DEL individual as RHD(+)/RHD(+) homozygote. It is concluded that the sensitivity of detecting D antigen by flow cytometry is similar to that of IAT, but lower than absorption/elution test. As for detecting weak D or partial D antigens, IAT is easier than flow cytometry; as for identifying DEL, the flow cytometry is not sensitive enough.
Adult ; Alleles ; Blood Donors ; Blood Grouping and Crossmatching ; Flow Cytometry ; methods ; Genotype ; Humans ; Phenotype ; Rh-Hr Blood-Group System ; blood ; genetics ; immunology

To identify HLA novel allele in Chinese Han individuals, an unknown HLA-A allele was detected by PCR-SSP and FLOW-SSO in Chinese Han individuals. Heterozygous sequence-based typing (SBT) showed that there were 3 differences compared with database in exon 2. Its anomalous patterns suggested the possible presence of either a novel A * 30 or a novel A * 24. To separate the two alleles and to determine whether the allele is novel, the HLA-A * 30 and HLA-A * 24 alleles were amplified separately by using a commercial kit for the single allele-specific sequencing strategy, and both alleles for exons 2 - 4 were sequenced according to the manufacturer' protocol. To prepare B-lymphoblastoid cell line of the novel HLA allele by using Epstein-Barr virus-infected B-lymphoblastoid cells in the peripheral blood. The results indicated that the sequencing results showed HLA-A alleles of the sample to be HLA-A * 240201 and a new A * 30 allele. The sequences of the new A*30 were identical to those of HLA-A * 300101 except for three nucleotide changes in exon 2: at nt 121 (A-->C), nt 123 (T-->C) and nt 126 (A-->G), resulting in an amino acid residue substitution from S (AGT) to R (CGC) at codon 17 and a synonymous substitution from G (GGA) to G (GGG) at codon 18. Immortalized B-lymphoblastoid cell line of the novel HLA-A * 3018 allele was achieved, the sequence of HLA-A * 3018 allele was submitted to GenBank and its accession number was DQ872509. In conclusion, the HLA-A * 3018 is a novel HLA-A allele and has been officially named HLA-A * 3018 by the WHO Nomenclature committee in August 2006 (HWS10004039).
Alleles ; Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; HLA-A Antigens ; genetics ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA

RHD gene has different alternative transcripts. This study was aimed to construct expression vector of normal mRNA, DEL9 and DEL89 transcripts from RHD gene. Total RNA was extracted from Rh(D) positive umbilical blood cells of newborn. Intact RhD cDNA, DEL9 and DEL89 transcripts were obtained by one-step and two-step RT-PCR, respectively. The obtained products were cloned into pCR4 TOPO sequencing vector for choosing the right transcript. RHD gene was amplified again from the sequencing plasmid DNA, and then subcloned into pcDNA3.1/V5-His TOPO expression vector; DEL9 and DEL89 were cloned into the expression vector directly. Gene sequence and direction were identified by sequencing. The results showed that the sequence and direction of target genes were right, thus these 3 different expression vectors were correctly constructed. It is concluded that expression vector is constructed from different transcripts of RHD gene, which lays a foundation for further exploring the membranous protein expression of Rh(D) antigen.
Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA

AIMTo compare the use of the suprapubic puncture method versus the transurethral method in pressure-flow studies in patients with benign prostatic hyperplasia.

METHODSTwenty-three men with benign prostatic hyperplasia underwent both suprapubic and transurethral pressure-flow studies during a single session. Standard pressure-flow variables were recorded in all patients with both methods, enabling calculation of obstruction using commonly used grading systems, such as the urethral resistance algorithm, the Abrams-Griffith (AG) number and the Schaer linear nomogram.

RESULTSThere were statistically significant differences between the methods in the mean values of maximum flow rate (P < 0.05), detrusor pressure at the maximum flow (P < 0.01), urethral resistance algorithm (P < 0.01), AG number (P < 0.01) and maximum cystic capacity (P < 0.01). Of the men in the study, 10 (43.5%) remained in the same Schaer class with both methods and 18 (78.3%) in the same AG number area. Using the transurethral method, 12 (52.2%) men increased their Schaer class by one and 1 (4.3%) by two. There were also differences between the suprapubic and transurethral methods using the AG number: 4 (17.4%) men moved from a classification of equivocal to obstructed and 1 (4.3%) from unobstructed to equivocal.

CONCLUSIONThe differences between the techniques for measuring intravesical pressure alter the grading of obstruction determined by several of the commonly used classifications. An 8 F transurethral catheter significantly increases the likelihood of a diagnosis of bladder outlet obstruction when compared with the suprapubic method.

Aged ; Humans ; Male ; Middle Aged ; Pressure ; Prostatic Hyperplasia ; physiopathology ; Punctures ; Urinary Bladder Neck Obstruction ; diagnosis ; Urinary Catheterization ; Urination ; physiology ; Urodynamics

Pressurized solvent extraction (PSE) is a new extraction technology which has been developed in recent years and widely used as sample analysis in environmental, food and pharmaceutical fields. Extraction technique is a key technology in quality control of Chinese herb. Conventional extraction techniques have been a bottleneck of blocking the development of quality control of Chinese herb. This article attempts to review the basic principle, methods, apparatus and main characteristics of PSE and its applications to quality control of Chinese herb.
Chemistry Techniques, Analytical ; methods ; standards ; Drugs, Chinese Herbal ; isolation & purification ; Plants, Medicinal ; chemistry ; Pressure ; Quality Control ; Solvents ; Technology, Pharmaceutical ; methods ; standards

OBJECTIVETo observe the effect platelet antigen modification by mPEG-SPA with different molecular masses.

METHODSPlatelet CD42a was modified by 5 kD and 20 kD mPEG-SPA, respectively, and the fluorescence intensity of CD42a was detect by flow cytometry and the three-dimensional structure of CD42a simulated to analyze the distribution of lysine in CD42a molecule.

RESULTSAfter platelet CD42a modification by 5 kD and 20 kD mPEG-SPA, the fluorescence intensity of CD42a decreased sharply by 85.54% and 88.65%, respectively, and multiple lysine regions were identified on the surface of CD42a molecule.

CONCLUSIONBoth 5 kD and 20 kD mPEG-SPA allow useful modification of platelet CD42a, but 20 kD mPEG-SPA is more advantageous than 5 kD mPEG-SPA.

Blood Platelets ; chemistry ; Humans ; Molecular Weight ; Platelet Glycoprotein GPIb-IX Complex ; chemistry ; Polyethylene Glycols ; chemistry ; Succinimides ; chemistry

OBJECTIVETo study the genetic feature of weak D type 15 allele (RHD845A) in a Chinese family.

METHODSRh D, C, c, E and e phenotypes of 4 members in a weak D type 15 family were tested by serological and polymerase chain reaction (PCR), D antigen was proven by indirect antiglobulin test. A pair of primers specific for RHD845A were designed, and a sequence specific primer-PCR (PCR-SSP) method was established to detect RHD845A allele in all family members. Subsequently the dual-tube PCR method was used to determine the RHD zygosity of 4 members.

RESULTSThe RHD845A allele existed in all 4 family members and the RHD zygosity test showed that all members were RHD +/RHD + homozygous. The parents and nephew possessed one normal RHD gene as RHD845A allele carriers, which caused RhD positive. The proband and his old-sister took two RHD845A alleles, which caused weak D phenotype.

CONCLUSIONThe proband is the weak D type 15 allele homozygous. The weak D type 15 gene is an ancestral allele, but not a mutation.

Adult ; Alleles ; Family Health ; Female ; Genotype ; Homozygote ; Humans ; Male ; Pedigree ; Point Mutation ; Polymerase Chain Reaction ; Rh-Hr Blood-Group System ; genetics