[Phenotype Types and Genetic Mutation Mechanism of Rhesus D Variant Individuals].

OBJECTIVE: To explore the phenotype types and genetic mutation mechanism of Rhesus D variant individuals. METHODS: Fouty-eight peripheral blood samples of pregnancies and blood donors who had been identified as Rhesus D variant by using routine serologic methods were collected from January 2013 to October 2015 in our center. The multiple ligation-dependent probe amplification(MLPA) was used to determine the RHD after genomic DNA had been extracted from the blood sample, then the data including gene copy number variations, point mutations, deletions and hybrid fusions were analyzed by GeneMarker software. All exons of blood sample RHD were amplified via PCR and analyzed by sequencing when its MLPA results were not in accordance with serologic results. Cloning and haplotype sequencing were performed if novel allele had been found. RESULTS: Rh phenotypes of the 48 samples were typed as following: 20 cases out of 48 were CcDee(41.7%, 20/48),12 cases were ccDEe (25%,12/48), 11 cases were CCDee(22.9%, 11/48), 5 cases were CcDEe (10.4%, 5/48), respectively. The MLPA analysis showed that 38 cases possessed only 1 variant allele(RHD zygosity was Dvd), while 10 cases possessed 2 variant alleles(RHD zygosity was DvDv). In Dvd type individuals, point mutations were found in 18 cases and RHD/CE hybrid fusions were found in 20 cases. In DvDv individuals, point mutations combined with RHD/CE hybrid fusions were found in 9 cases, deletion combined with RHD/CE hybrid fusions were found in 1 case. Variant alleles analysis basing on MLPA showed that 14 cases were weak D 15 and 22 cases were RhD VI type 3, however, the variant alleles were not identified in 7 cases due to lack of detecting probes and were identified via sequencing analysis. Two novel mutations, 79-81delCTC and 689G>A were also certificated by sequencing in 2 cases. CONCLUSION: CcDee is the major Rh phenotype in RhD variants, weak D 15 and RhD VI type 3 are the main serologic type of RhD variants, point mutation and RHD/CE hybrid fusions are main molecular mechanism for RhD variant phenotype. Besides, 79-81delCTC and 689G>A are two novel alleles.

[Immunoserology and RHD Genotype Analysis of DVI Type 3 Genotype Pregnant Women with Anti-D].

OBJECTIVE: To performe the immuneserological and RHD Genotype analyses for DVI type 3 genotype pregnemt women with anti-D. METHODS: RhD blood type of this pregnant women was identified by common serological methods, then the blood group specific antibodies was screened and identified; the polymerase chain reaction-sequence specific primer(PCR-SSP) was used to identify the pregnant women's RHD genotype; RhD blood group for the pregnant women, her spouse and daughter was genogrouped and genetically analyzed by multiplex ligation-dependent probe amplification(MLPA). The heredity of this family was analyzed finally. RESULTS: The titer of IgG anti-D in the pregnant woman serum was 1:8; the PCR-SSP showed that the 3rd to 6th exons of RHD gene were missing in the pregnant woman. the genotype of pregnant woman was identified as DVI type 3; the MLPA analysis showed that this pregnant women owned only one RHD allele with 3rd to 6th exons missed, and her genotype was identified as CDVIe/cde; her spouse was identified as CDe/CDe homozygous genotype, and her daughter as CDe/CDVIe. CONCLUSION: Accurate identification of RhD blood type is of great significance for a safe and effective clinical blood transfusion strategy, and for taking appropriate measures to prevent hemolytic disease of newborn (HDN) at women childbearing age.

[Affinity Detection and Quantification of Receptors of TRAIL Based on Their Thermostability].

OBJECTIVE: To develop a way for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors quantification in cancer via its' thermostability. METHODS: Endougenous alkaline phosphatase (AP) activity and denaturation temperature of pancreatic cancer cell lines AsPC-1 and Capan-2 were detected. Boiling treated recombinant protein death receptor 5 (DR5), named DR5 AP, as well as pancreatic cancer cells lines AsPC-1 and Capan-2 were incubated with AP-tagged TRAIL (AP-TRAIL), and then reacted with Reagent A and Reagent S, the substrate of AP, to quantitive and in site detection of the receptor. RESULTS: The endougenous AP activity of pancreatic cancer cells lines AsPC-1 and Capan-2 could not be totally inactivated by incubated at 65 °C, thus inhibited the detection of TRAIL receptor, but the activity was dramatically decreased after treated with boiling water, whereas the DR5-AP was thermal stable. The surface receptor of AsPC-1 and Capan-2 could be recognized and bound by AP-TRAIL after treated at 100 °C, the readings were 2. 210±0. 393 and 2. 027±0. 019. CONCLUSION: The TRAIL receptors are thermostable and this may provide a better diagnosis and prognosis of cancer as well as personalize cancer therapy.

[Serological and genetic study of a pedigree featuring a rare p phenotype].

OBJECTIVE: To explore genetic background of a pedigree with a rare p phenotype from Guangdong province. METHODS: The rare p phenotype was identified by a conventional serologic method. With genomic DNA of proband and family members extracted, exon 3 of alpha-(1,4)galactosyltransferase (A4GALT) gene was amplified with PCR and analyzed by direct sequencing. The mutation found in the pedigree was screened in a normal population using direct sequencing. RESULTS: The proband and 4 family members with the rare p phenotype have all carried a point mutation c.100G>A (p.Val34Ile) in combination with a deletion-insertional mutation c.418_428del11ins34(p.Gln139Trpfs*72), which renders a compound mutation of A4GALT gene. One family member with P2 phenotype has carried a same heterozygous mutation. Of the 100 healthy donors, 5 have carried a heterozygous point mutation c.100G>A, and none carried the deletion-insertional mutation c.418_428del11ins34. CONCLUSION: The rare p phenotype of the pedigree has resulted from a compound mutation of the A4GALT gene, which is in keeping with a recessive inheritance pattern of the p phenotype.

[Human platelet lysates promotes the proliferation of mesenchymal stem cells in vitro].

OBJECTIVE: To investigate the effect of human platelet lysates (HPL) obtained from platelet-rich plasma on the proliferation and biological characteristics of human mesenchymal stem cells (MSCs) in vitro. METHODS: HPL was obtained by repeated freeze-thawing of human plateletes, and the MSCs separated by density gradient centrifugation from 6 donors were expanded in medium supplemented with 10% fetal bovine serum (FCS) or HPL at different concentrations. The optimal concentration of HPL for cells culture was determined according to the cell proliferation kinetics. The cultured MSCs were characterized for their proliferation, cell phenotype, and cell cycle distribution. RESULTS: The HPL-supplemented medium contained 4 essential growth factors for the growth of MSCs, namely platelet-derived growth factors (0.53∓0.06 ng/ml), basic fibroblast growth factor (37.5∓4.31 pg/ml), insulin-like growth factor-1 (0.15∓0.06 mg/ml) and transforming growth factor (5150∓463 pg/ml). Cultured in the presence of HPL at the optimal concentration of 7.5%, the MSCs displayed a spindle-shaped fibroblast-like morphology without obvious changes in the proliferation activity till passage 8 (P>0.05), similar to those of cells in FCS-supplemented culture medium. Flow cytometry and cell cycle analysis revealed no differences in the phenotypes or cell cycle distribution between the cells cultured in the presence of 7.5% HPL and 10% FCS. CONCLUSION: The culture medium supplemented by 7.5% HPL can promote the expansion of human MSCs and maintain the basic biological characteristics of the cells.

[Study of the platelet GP specific antibodies and HLA antibodies expression in platelet transfusion refractoriness patients].

OBJECTIVE: To investigate the correlation between the platelet GP specific antibodies/HLA antibodies and platelet transfusion refractoriness (PTR). METHODS: Sixty-five patients with PTR were selected in this study and were genotyped for HLA-A and HLA-B as well as HPA systems by standard PCR-SSP assays. The platelet GP specific antibodies and HLA antibodies in serum and platelet elution were tested with a solid phase ELISA. RESULTS: The HLA-A/B antigens and the frequencies of HPA-1, 2, 4, 5, 6, 9, 15 antigens in PTR patients had no difference from those in healthy donors. The freguencies of HPA-3a and 3b were 0.65 and 0.35, respectively. There was statistical difference between the 65 PTR patients and the healthy donors in HPA-3 freguencies (P < 0.05). Twenty-four patients (36.9 %) only expressed HLA antibodies, and 14 (21.5%) expressed HLA and platelet GP specific antibodies. The highest expression of anti-HLA-A/B specific antibodies was -A*9(46.2 %)/-B*40(33.6%), respectively. In serum, GPIIb/IIIa was expressed (26.2%), followed by GPIa/IIa (21.5 %). In platelet elution, GPIIb/IIIa was expressed of 41.5% and GPIb/IX 41.5%. Pedigree study was carried out for 2 patients. The results showed that the platelet GP specific antibody/HLA antibody developed in PTR patients was highly related to the mismatch with the platelet antigen/HLA antigen in their parents. CONCLUSION: The expressions of the HLA and platelet GP specific antibodies are the most important reason in PTR, it's meaningful to explore the correlation between PTR and HLA and HLA-A/B antigen in guiding platelet transfusion.

[Expression of anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in idiopathic thrombocytopenic purpura].

In order to investigate the expression of the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in idiopathic thrombocytopenic purpura (ITP), 45 patients with ITP were selected in this study. An easy PCR-SSP assay was used to detect single-nucleotide polymorphisms or deletion in HPA and HLA systems. The anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in plasma or platelet eluate were tested with a solid phase ELISA. The results indicated that the anti-platelet glycoprotein specific antibodies were detected in plasma or platelet eluate of 45 patients, among which anti-GPIIb/IIIa/and anti-GpIb/IX were most common. Both the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies were found in plasma of 11 patients. Pedigree investigation in 2 patients (case 37 and case 40) was carried out, the results showed that anti-platelet glycoprotein specific antibodies and anti-HLA antibodies detected in 2 patients closely related to incompatibility with platelet antigens and HLA antigens in parents. In conclusion, the results suggested that detection of the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in plasma or platelet eluate in combination with investigation of clinical manifestation of patients is important for diagnosis of idiopathic thrombocytopenic purpura.

[Biological appraisal of human bone marrow mesenchymal stem cells during ex-vivo expansion].

This study was aimed to investigate the characteristics of human bone marrow mesenchymal stem cells during ex-vivo expansion, MSCs were isolated from human bone marrow. At each passage, the characteristics of proliferation kinetics, osteogenic and adipogenic differentiation potential were analyzed, and cell morphology, surface markers were investigated as well. The karyotype analysis was done in different passage cells. The infection HIV, HCV, HBV and TP were detected by ELISA. Mycoplasma contamination in vitro was detected by PCR method. HLA-SBT was used to reanalyze the results of HLA antigens and alleles. STR genetic loci were detected by PCR in the MSC1, MSC2, MSC3 and MSC4. The results indicated that the proliferative ability and osteogenic potential decreased with the increase of passage number during culture expansion. The multiple differentiation potential of MSCs was maintained during their life span. Karyotype analysis showed that MSCs from 4 groups before passage 8 were normal. The expression of CD29, CD44, CD105, CD166 and CD73 were positive. The expression of CD14, CD34, CD45, CD80, CD86 were all negative. SBT was used to identify HLA-A, B, Cw, DRB1, DRPB1, DQ alleles in the MSC1, MSC2, MSC3, MSC4. The genetype of STR in the MSC1, MSC2, MSC3, MSC4 was different. MSC 3 was examined by TP-ELISA to confirm the infectious disease of TP. MSC2 was contaminated by mycoplasma at passage 5. It is concluded that culture expansion causes MSCs to gradually lose their stem cell properties. During ex-vivo expansion of MSCs, the osteogenic differentiation potential is decreased. MSCs before passage 8 can be a valuable subject for basic research and clinical application.

[Genotyping of ABO loci in para-Bombay type individuals].

To study the molecular genetic basis of ABO alleles in para-Bombay type individuals, samples from five para-Bombay type individuals identified by serologic tests including absorption-elution tests, saliva neutralizing or inhibitor substances tests, were genotyped by using PCR-SSP based ABO genotyping. Exon 6 and exon 7 at the ABO locus for all 5 samples were sequenced. The results showed that the ABO genotypes of five para-Bombay samples were A102B1, A102B1, A102O1, A102B1, B1O1 respectively, the direct DNA sequencing results were in accordance with the results genotyped by PCR-SSP method, No novel nucleotide mutation was found at the exon 6 and exon 7 of ABO gene. In conclusion, the ABO genotyping assay by PCR-SSP provide a simple, rapid and accurate method for determining the ABO type of para-Bombay cases.