Molecular and Family Analyses of a Novel RHD1058G>C Allele in a Chinese RhD Population.

BACKGROUND: Rh(D) phenotype in a sample from a 19-year-old female patient showed weak positivity (1+). A follow-up sample was requested to further define the Rh(D) phenotype, her Rh(D) phenotype was tested by using another reagent, Rh(D) phenotype still showed weak reactivity (1+), RhCcEe phenotype was Ccee. METHODS: Seven samples from the family members of the proposita were received. The RhDCcEe phenotypes were typed by the microcolumn gel card and the unexpected antibodies were assayed by indirect anti-human globulin test (IAT). Genomic DNA was extracted from the blood sample and the novel RHD1058G>C allele was detected through an established sequence-specific primer PCR (PCR-SSP), RHD exons 1 - 10 were sequenced afterward by exon-specific amplification. The distribution of RHD1058G>C allele and RHD weak positive phenotype were investigated in the pedigrees. RESULTS: The unexpected antibodies all were negative in the family members. The novel RHD1058G>C allele was found in the proposita, her father, and grandfather. Five family members were detected serologically with the common Rh(D)-positive phenotypes either as homozygote of RHD/RHD or heterozygote of RHD/RHd. Two family members were detected as weak D phenotypes in accordance with the genotyping results by PCR-SSP, and both of them have a D1058Ce haplotype and a dce haplotype. One member, her father, was tested common Rh(D)-positive with D1058Ce haplotype and a Dce haplotype. CONCLUSIONS: These data allow us to describe the characteristics of the weak D phenotype with a novel c.RHD-1058G>C allele, which may be partial D and increase the risk of RHD alloantibody. The novel RHD1058G>C allele was inherited in three generations in a family rather than spontaneous mutation in an individual.

A Rare Heterozygote with a Novel IVS-â ¡-786 (T>A) Mutation on ß-Globin Gene in a Patient with Thalassemia.

BACKGROUND: Thalassemia is an inherited hemolytic blood disease, whose pathogenesis is an imbalance in the expression of hemoglobin. We report a case of a rare ß-globin gene intron mutation for thalassemia patient. METHODS: The blood routine test was performed with an automatic blood cell analyzer. Hb analysis was conducted by hemoglobin (Hb) analyzer. The common ß-thalassemia and α-thalassemia gene mutations were detected by Gap-PCR and fluorescence PCR melting curve, and the rare ß-thalassemia gene mutations were detected by DNA sequencing. RESULTS: A rare heterozygous mutation of ß-globin gene IVS-II-786 (T>A) was found in this case. Blood routine analysis showed the following values: Hb 92 g/L, RBC 4.1 x 1012/L, MCV 74.10 fL, MCH 22.4 pg, MCHC 303 g/L, HCT 0.304 L/L, and RET-He 22.7 pg. Hemoglobin analysis showed values of HbA2 2.2% and HbF < 2% by automatic capillary electrophoresis. The results of gene analysis and DNA sequencing showed that the ß-globin gene IVS-II-786 (T>A) mutation was heterozygous. CONCLUSIONS: The heterozygote of ß-globin gene IVS-II-786 (T>A) mutation was detected for the first time, and the clinical manifestation was moderate anemia. Hemoglobin analysis indicated that the level of HbA2 was decreased. This mutation is relatively rare and easy to misdiagnose in clinical practice. It will provide a new type of evidence and guidance for genetic counseling and clinical treatment of beta thalassemia.

A Novel RHCE*cE (c.827C>A) Allele, Containing the Single-Nucleotide Change, Encodes Altered c/E Antigens.

The RH blood group system is the most complex with over 50 antigens. So far over hundreds of RhCE variant alleles have been described resulting in weakened and/or partial expression of RhCE antigens [1], some variant Rh phenotypes are caused by exchange of genetic material between the RHD and RHCE genes, resulting in many hybrid genes, other phenotypes result from missense mutations. Variant alleles encode altered phenotypes with either weakened antigens, lacked antigens, or unexpected antigens. Besides, the mutation of RH blood group genes may lead to the changes of Rh antigen epitopes. RHCE gene mutations or polymorphisms may bring about altered RH antigens in quality and quantity [2]. Serologic weaknesses or discrepancies are regularly faced by blood transfusion laboratories, and molecular background explaining this feature can be precisely characterized only by the molecular biological methods.

[Identification of RhCcEe Mixed Visual Field in Patients with Regular Blood Transfusion and Efficacy Analysis of the Matched Transfusion].

OBJECTIVE: To explore the feasibility of RhCcEe blood group antigen mixed visual field identification in patients with regular blood transfusion, to follow up and evaluate the efficacy of matched transfusion and its clinical significance. METHODS: RhCcEe genotyping for 142 patients with regular transfusion in our hospital was carried out by PCR-SSP method. According to the results of genotyping, 48 patients voluntarily selected the continuous transfusion of RhCcEe matched red blood cells, 46 patients received random blood transfusion (RhCcEe mismatched transfusion), 42 patients received partial RhCcEe matched transfusion (unable to provide fully matched RhCcEe donors each time), and 6 patients' blood transfusion data were lost. After 3-6 months of the RhCcEe matched transfusion, all patients were tested by RhCcEe microcolumn gel card and compared with the results before RhCcEe matched transfusion. The positive rates of alloantibodies, DAT and the percentage of red blood cell invalid transfusion were followed up and evaluated for the above-mentsioned 3 types of regular transfusion patients in the past 5 years. RESULTS: Out of the 48 patients who underwent conti-nuous RhCcEe matched transfusion, only 1 case showed stratification, the remaining 47 cases had clear gel card results without stratification, suggesting that PCR-SSP genotyping was feasible. In addition, another 42 patients who could not receive RhCcEe matched transfusion each time and 46 patients with random blood transfusion were found to have a mixed vision phenomenon again. but the results was still difficult to confirm the results. For the transfusion results in the past 5 years, follow-up analysis showed that there were 1 case alloantibody (anti-Jka) (1/48) , 1 case of DAT positive (1/48) and 2 cases of invalid transfusion (2/48) in the RhCcEe matched transfusion group; 7 cases of alloantibodies (3 anti-E, 1 anti-E+anti-c, 1 anti-C, 1 anti-M, 1 anti-Fya) (7/46), 6 case of DAT positive (6/46) and 9 case of invalid transfusion (9/46) in the random transfusion group; 6 cases of alloantibodies (1 anti-E, 1 anti-E+autoantibody, 1 anti-C, 1 anti-c, 1 anti-M and 1 other antibody) (6/42) and 7 case of DAT positive (7/42) and 8 case of invalid transfusion (8/42) in the partial RhCcEe matched transfusion group. The statistical analysis showed that the positive rate of alloantibodies and the invalid infusion rate of RBC in each group were significant differences between RhCcEe matched transfusion group and the random transfusion group as well as betwen Rhce fe matched transfusion group and the partial matched transfusion group（P＜0.05）, but there was no statistical difference between the random transfusion group and the partial matched transfusion group（P>0.05）. CONCLUSION: PCR-SSP genotyping technique can be used to detect RhCcEe mixed vision in patients with regular blood transfusion. Continuous RhCcEe matched transfusion can effectively prevent the occurrence of alloimmunization, and improve the clinical transfusion efficacy and safety of the patients with regular blood transfusion, which has very important clinical significance.

[Screening and Identification of Blood Group Alloantibody by Surface Plasmon Resonance Technique and Its Preliminary Application].

OBJECTIVE: To investigate the feasibilily of screening and identifying the red blood cell type alloantibodies by means of surface plasman resonance(SPR) technique so as to provide a new method for detecting the transfusion compatibility of red blood cells. METHODS: The RBC antigens for screening the alloantibody were fixed on the SPR chip surface by means of amino coupling method; the analysis conditions of SPR chip were optimized and then the control serum with RBC blood group antibody positive was detected; the performance of SPR chip for detection of serum was analysed; the consistance of rusults detected by SPR technique and microcolum agglutination for clinieal samples of 129 thalasstmia patients with history of lone-term blood transfusion were compared; at the same time, the blood group amtibodies in 7 patients with blood group antibody positive were identified before blood transfusion by using SPR chip so as to select the RBC antigen compatible blood for transfusion; and the efficacy of RBC transfusion was followed up and evaluated. RESULTS: The repeatability, sensitivity and specificity of SPR chip technique for detecting the blood group alloantibodies all were better. The SPR technique and microcolumn agglutination method were not significant different for screening blood group alloantibodies (χ2 = 0.333, P＞0.05), and the overall consistency was 97.2%; the results of SPR technique in 7 patients with positive blood group antibodies were as follows: 3 cases with anti-E, 1 case anti-M, 1 case anti-C, 1 case anti-Jka and 1 case autoantibody, which were consistent with the results of microcolumn agglutination tests, and the compatible red blood cells were selected for transfusion, of which the infusion of 6 cases was effective. In only 1 case the infusion was ineffective because of autoantibody. CONCLUSION: For screening and identification of blood group alloantibodies, the performance of SPR chip technique is equivalent to the micro-column agglutination, but the procedure of SPR technique is simpler, faster and high-throughput and label-free, which can meet the basic requirements for rapid screening and identification of blood group alloantibodies before transfusion of red blood cells.

Surface plasmons excited by multiple layer grating.

The surface plasmons that are excited by the multiple layer grating structures on the gold thin film are studied using the finite-difference time-domain method in this paper. The structure parameters' effects on the coupling enhancement of surface plasmons are examined, and the structure design guidelines are given. It is found that the distance between the grating layers and the distance between the gratings and gold thin film are the key structure parameters for better cavity resonances. To have the stronger field enhancements of the excited surface plasmons for the multilayer grating structures, it is found that the width of the gratings should be smaller for the lower grating layers. The multiple layer gratings with proper structure designs can have better performances than single layer grating structure because the cavity effects can enhance the light coupling and more light can be coupled into the surface plasmons by more layers of grating. It is found that the maximum electric field intensity for five layer grating structures can be 163% of the case of the single layer grating structure in our simulations.

Rh-Matched Transfusion through Molecular Typing for ß-Thalassemia Patients Is Required and Feasible in Chinese.

BACKGROUND: Molecular typing for RHCE blood group alleles has been established in many countries for patients and blood donors. In the Chinese literature nearly 80% of transfused patients with alloimmunization have antibodies specific for antigens of the Rh blood group system. We investigated if it is feasible to match packed red blood cells (RBCs) for Chinese ß-thalassemia patients by RHCE genotyping. METHODS: In this study, 481 patients with ß-thalassemia were enrolled. They were genotyped for RHCE alleles by a simple PCR method with sequence-specific primers (PCR-SSP). Among these patients, 203 continuously received RBCs of the identical Rh subgroups according to the genotyping results for at least 3 months. Subsequently, their phenotypes were tested through a micro-column gel card method. For validation purposes, 400 donors were serologically typed with the same technology, of which 164 were genotyped too. Finally, the C, c, E, and e frequencies and the feasibility of the simple genotyping method were analyzed. RESULTS: All patients showed mixed-field agglutination in the Rh subgroup gel cards before the same Rh subgroups in blood donors were selected for blood transfusion. The results, however, lacked mixed-field agglutination in all 203 cases after transfusion with RBC concentrates selected for the patient's C, c, E, and e antigens for at least 3 months. The genotyping results of 164 donors were all consistent with the serological results. Whole coding regions of RHCE were sequenced in 7 individuals with weak c, E, or e antigens. In only one sample we observed a 1059G>A nucleotide mutation coding for a truncated RhCE polypeptide (GenBank KT957625), in the other 6 samples no sequence variant was found. Both patients and donors were predominantly CcEe and CCee, with a prevalence of 55.3% and 24.9% for patients or 49.3% and 31.3% for donors, respectively. It revealed that about 80% of Chinese could receive Rh-matched RBCs easily. CONCLUSION: A simple RHCE genotyping technique is safe enough for Rh-matched transfusion of ß-thalassemia patients in Chinese Han.

Development of a Strategy Based on the Surface Plasmon Resonance Technology for Platelet Compatibility Testing.

BACKGROUND: This study was aimed to establish a novel strategy based on the surface plasmon resonance (SPR) technology for platelet compatibility testing. METHODS: A novel surface matrix was prepared based on poly (OEGMA-co-HEMA) via surface-initiated polymerization as a biosensor surface platform. Type O universal platelets and donor platelets were immobilized on these novel matrices via amine-coupling reaction and worked as a capturing ligand for binding the platelet antibody. Antibodies binding to platelets were monitored in real time by injecting the samples into a microfluidic channel. Clinical serum samples (n = 186) with multiple platelet transfusions were assayed for platelet antibodies using the SPR technology and monoclonal antibody-immobilized platelet antigen (MAIPA) assay. RESULTS: The novel biosensor surface achieved nonfouling background and high immobilization capacity and showed good repeatability and stability after regeneration. The limit of detection of the SPR biosensor for platelet antibody was estimated to be 50 ng/mL. The sensitivity and specificity were 92% and 98.7%. It could detect the platelet antibody directly in serum samples, and the results were similar to MAIPA assay. CONCLUSIONS: A novel strategy to facilitate the sensitive and reliable detection of platelet compatibility for developing an SPR-based biosensor was established in this study. The SPR-based biosensor combined with novel surface chemistry is a promising method for platelet compatibility testing.

Linking arsenite- and cadmium-generated oxidative stress to microsatellite instability in vitro and in vivo.

Mismatch repair (MMR) corrects replicative errors and minimizes DNA damage that occurs frequently in microsatellites. MMR deficiency is manifested as microsatellite instability (MSI), which contributes to hypermutability and cancer pathogenesis. Genomic instability, including MSI and chromosomal instability, appears to be responsible for the carcinogenesis of arsenic and cadmium, common contaminants in our environment. However, few studies have addressed arsenic- or cadmium-induced MSI, especially its potential link with arsenic- or cadmium-generated oxidative stress, due to the lack of quantifiable MSI assays and cost-effective animal models. Here, using a dual-fluorescent reporter, we demonstrate that sub-lethal doses of cadmium or arsenite, but not arsenate, increased the MSI frequency in human colorectal cancer cells. Arsenite- and cadmium-induced MSI occurred concomitantly with increased levels of reactive species and oxidative DNA damage, and with decreased levels of MMR proteins. However, N-acetyl-l-cysteine (NAC) suppressed arsenite- and cadmium-induced MSI and oxidative stress while restoring the levels of MMR proteins in the cells. Similarly, MSI was induced separately by arsenite and cadmium, and suppressed by NAC, in zebrafish in a fluorescinated PCR-based assay with newly-developed microsatellite markers and inter-segmental comparisons. Of five selected antioxidants examined, differential effects were exerted on the MSI induction and cytotoxicity of both arsenite and cadmium. Compared to MMR-proficient cells, MMR-deficient cells were more resistant to arsenic-mediated and cadmium-mediated cytotoxicity. Our findings demonstrate a novel linkage between arsenite-generated and cadmium-generated oxidative stress and MSI induction. Our findings also caution that antioxidants must be individually validated before being used for preventing arsenite- and cadmium-induced MSI that is associated with cancer development.

[RH Factor and Clinical Transfusion Effectiveness for ß-Thalassemia Children with Long-Term Blood Transfusion].

OBJECTIVE: To investigate the irregular antibody production and its relationship with Rh factor genotypes and the loci of thalassemia gene mutations for the ß-thalassemic children with long-term transfusion, so as provide experimental basis for clinical safe and effective transfusions for thalassemic children. METHODS: The peripheral blood from 246 children with ß-thalassemia was collected in our hospital; the extraction of genomic DNA and Rh factor (C/c, E/e) genotypes were assayed by PCR-SSP method, the irregular antibodies were screened and identified by serological method, the genotypes for thalassemia and gene mutations were analysed by PCR-RD method. RESULTS: The genotypes of Rh factors classified by PCR- SSP in the 246 cases of ß-thalassemia children were as follws: Ce/Ce (143/246, 58.1%), CE/ce (59/246, 24%), cE/cE (14/24, 5.7%), Ce/ce (12/246, 4.9%); The positive rate of irregular antibody was 7.7% (19/246), including anti-E (7/19), anti-c (5/19), anti-C (2/19), anti-E and anti-c (2/19), anti-e (1/19), anti-D (2/19); Of the 19 cases with positive irregular antibody, the genotypings of Rh factor were: Ce/Ce (11/19), CE/ce (2/19), cE/cE (2/19), Ce/ce (2/19), cE/ce (2/19); the gene mutations location of thalassemia for 19 cases with positive irregular antibody: CD41-42M (13/19), CD71-72M (2/19), IVS-II-654M (3/19), -28M (1/19). CONCLUSION: The irregular antibody production for ß-thalassemic children with long-term transfusion may have some relevance with Rh factor genotypes and thalassemia genetic mutations. This study possesses a certain significance for effective prevention of RBC alloimmune response of ß-thalassemia children and improvement of efficacy and safety of clinical transfasion blood.

A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress.

The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H2O2-generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2-generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.

[Correlation of Breg with CD4(+)T cells of peripheral blood in patients with CITP and its clinical significance].

This study was aimed to detect the level of the peripheral blood Breg and CD4(+) T cell subgroups in patients with chronic idiopathic thrombocytopenic purpura (CITP) before and after therapy, and to analyse the charge of related cytokines and their correlation, to explore their roles in the pathogenesis of CITP. A total of 35 CITP cases were taken as the research group and 35 healthy persons were served as the control group. The peripheral blood mononuclear cells (PBMNC) were separated, the percentages of Th1, Th17, Th22 and Breg cells were detected by flow cytometry before and after treatment of glucocorticoid, and the IFN-γ, IL-17, IL-22 and IL-10 levels from PBMNC culture supernatant also were determined by ELISA. The results showed that there was significant difference as compared with the healthy controls, the proportion of peripheral blood Th1, Th17, Th22 cell subgroups all increased in CITP patients before treatment with glucocorticoid, the regulatory B cells (Breg) ratio was reduced, the differences had statistical significance (P < 0.05), but the differences were no statistically significant after treatment with glucocorticoid (P > 0.05). The levels of IFN-γ, IL-17, IL-22 from culture supernatant all increased in CITP patients before treatment, the level of IL-10 was lower than that of the healthy control, the difference was statistically significant (P < 0.05), but the there were no statistically significant differences after treatment (P > 0.05). There were positive correlation between the Breg cells and IL-10 expression in CITP patients (P < 0.05), the Breg cells and Th1, Th17, Th22 cells showed a negative correlation, IL-10 and IFN-γ, IL-17, IL-22 levels also showed a negative correlation. It is concluded that the down-regulation of regulatory B cells proportion and the IL-10 level may participate in the mechanism of CD4(+) T cell immunity disorder in CITP, which can provide new targets and ideas for the clinical immune regulation therapy.

Evolution of heavy metals in municipal solid waste during bio-drying and implications of their subsequent transfer during combustion.

Bio-drying has been applied to improve the heating value of municipal solid waste (MSW) prior to combustion. In the present study, evolution of heavy metals in MSW during bio-drying and subsequent combustion was studied using one aerobic and two combined hydrolytic-aerobic scenarios. Heavy metals were concentrated during bio-drying and transformed between different metal fractions, namely the exchangeable, carbonate-bound, iron- and manganese-oxides-bound, organic-matter-bound and residual fractions. The amounts of heavy metals per kg of bio-dried MSW transferred into combustion flue gas increased with bio-drying time, primarily due to metals enrichment from organics degradation. Because of their volatility, the partitioning ratios of As and Hg in flue gas remained stable so that bio-drying and heavy metal speciation had little effect on their transfer and partitioning during combustion. In contrast, the partitioning ratios of Pb, Zn and Cu tended to increase after bio-drying, which likely enhanced their release potential during combustion.

Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC)-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of caspase-3, mitochondria dysfunction and nitric oxide (NO) in human osteogenic sarcoma U-2 OS cells.

Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, have been shown to exhibit antineoplastic ability against many human cancer cells. In this study, we found that exposure of human osteogenic sarcoma U-2 OS cells to BITC and PEITC led to induce morphological changes and to decrease the percentage of viable cells in a time- and dose-dependent manner. BITC and PEITC induced cell cycle arrest at G2/M phase at 48 h treatment and inhibited the levels of cell cycle regulatory proteins such as cyclin A and B1 in U-2 OS cells but promoted the level of Chk1 and p53 that led to G2/M arrest. BITC and PEITC induced a marked increase in apoptosis (DNA fragmentation) and poly(ADP-ribose)polymerase (PARP) cleavage, which was associated with mitochondrial dysfunction and the activation of caspase-9 and -3. BITC and PEITC also promoted the ROS production in U-2 OS cells and the N-acetylcysteine (NAC, an antoxidant agent) was pretreated and then treated with both compounds which led to decrease the levels of ROS and increase the cell viability. Interestingly, BITC and PEITC promoted the levels of NO production and increased the iNOS enzyme. Confocal laser microscope also demonstrated that BITC and PEITC promoted the release of cytochrome c and AIF, suggesting that both compounds induced apoptosis through ROS, caspase-3 and mitochondrial, and NO signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC and PEITC-caused growth inhibition, G2/M arrest, and apoptotic death of U-2 OS cells.

Dietary effect of Antrodia Camphorate extracts on immune responses in WEHI-3 leukemia BALB/c mice.

Antrodia camphorata has been recognized to be a traditional Chinese medicine for abdominal pain, diarrhea, and to protect against hepatitis virus infection. Several ingredients derived from A. camphorata possess various pharmacological and biological activities such as antioxidant and anticancer. In this study, its ability to promote immune responses and to exhibited antileukemia activity in WEHI-3 leukemia BALB/c mice were investigated. The results indicated A. camphorata significantly prolonged the survival rate and prevented the body weight loss in leukemia mice. Four mg/kg of A. camphorata treatment significantly decreased the weight of the spleen. Both doses (2 and 4 mg/kg) of A. camphorata did not affect Mac-3 marker in leukocytes. However, the 4 mg/kg of A. camphorata decreased the levels of CD11b and both doses of treatment increased CD3 and CD19. With lipopolysaccharide stimulation, the 4 mg/kg of A. camphorata promoted the significant proliferation of leukocytes; but with concanavalin A stimulation, both doses promoted the significant proliferation of leukocytes. YAC-1 target cells were killed by NK cells from the mice after treatment with A. camphorata at 4 mg/kg in target cells at a ratio of 50:1. The percentage of macrophages with phagocyted at A. camphorata treatment increased, and these effects were in dose-dependent manners.