Expression Analysis of miRNA Profiles in Apheresis Platelets during Storage / 中国实验血液学杂志

OBJECTIVE@#To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage.@*METHODS@#The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value < 0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR).@*RESULTS@#By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P>0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing.@*CONCLUSION@#The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.

Effects of Apheresis Platelets Treated with Vitamin BPhotochemical Technology on the Release of White Blood Cells- and Platelet-Derived Cytokines during Storage / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate whether vitamin Bphotochemical pathogen reduction technology(PRT) treatment may lead to increase white cell- and platelet- derived cytokines release from platelets during storage.</p><p><b>METHODS</b>Sixty milliliters of leukodepleted apheresis platelets were collected from 20 healthy donors, then were divided into 2 parts: one part (30 ml) remained untreated to serve as control, while the other part was treated with vitamin B-UVB photo-chemical technology as experimental group. During 7 d of storage under standard blood bank conditions, platelet coun-ting (PC), platelet distribution width (PDW), mean platelet volume (MPV), white cell-derived cytokines (IL-1β, IL-2, IL-6, IL-8, TNF-α and IFN-γ) and platelet-derived cytokines (CCL3, CCL5, TGF-β-1 and PF4), P-selectin and phosphatidyl serine (PS) were analyzed on day 1, 3, 5 and 7 of storage, respectively.</p><p><b>RESULTS</b>No signi-ficant differences were observed on PC, PDW and MPV between the experimental and control groups, respectively. The higher levels of platelet-derived cytokines were detected and reached a plateau after 5-7 days of storage, and the cyto-kines showed significant increase in experimental group compared with the control group. PS expression increased signi-ficantly in experimental group as compared with control group on day 3, 5 and 7 of storage, respectively. The accumula-tion of P-selectin was significant higher in experimental group than that in control group on day 5 and 7 of storage (P<0.05). The white cell-derived cytokines were not elevated by PRT treatment during 7 days of storage.</p><p><b>CONCLUSION</b>The PRT-treated platelets are the main source of released cytokines during storage of PRT treatment. The levels of platelet-derived cytokines reach a plateau after 5-7 days of storage, most likely due to accelerated platelet activation and apoptosis.</p>

Identification of a novel HLA allele, HLA-DRB1*03:80, by sequencing-based typing / 中国实验血液学杂志

This study was aimed to identify a novel HLA-DRB1 allele from a Chinese potential hemopoietic stem cell donor of Northeast China. A rare HLA-DRB1 allele was initially detected by Luminex PCR-SSO typing, then the sample was sequenced by sequence-based typing (SBT) and the alignments of sample's alleles was identified by single allele-specific sequencing strategy. The results revealed the existence of a new allele which differs from the closest matching allele DRB1*03:06 by a single nucleotide substitution at position 239, where C→G in exon 2, resulting in an amino acid exchange from Thr to Arg at codon 51. It is concluded that a novel allele has been confirmed and its name DRB1*03:80 is officially assigned by the WHO Nomenclature Committee in February 2012.

Effects of N-Arachidonoylethanolamine on the quality of platelets stored in M-sol platelet preservative solution in vitro / 中国实验血液学杂志

This study was purposed to investigate the effects of N-Arachidonoylethanolamine (ANA) on the quality of platelets (Plt) stored in Plt M-sol preservative solution at 22 ± 2°C. Samples taken from collecting apheresis Plt by the Amicus instrument and splited into two equal parts were stored in Plt M-sol preservative solution on a shaker at 22 ± 2°C. Different working concentrations of ANA (from 0.1 to 50 µmol/L) were then added into one part of stored Plt as the experimental group, the other without ANA was used as the control group. The viability of Plts stored at 22 ± 2°C for 7 days was evaluated by MTT colorimetric assay. The most effective concentration of ANA was selected and added to the subsequent experimental group. Plt count (BPC), mean Plt volume (MPV), Plt distribution width (PDW), phosphatidyl serine (PS) and soluble P-selectin were detected on the 1(st), 5(th), 7(th), 9(th) and 11(th) day of storage. The results showed that the most effective working concentration of ANA was 0.5 µmol/L, which showed significant increasing Plt viability (91.23 ± 5.44%) compared to the control group (62.54 ± 4.79%). Thus, ANA concentration at 0.5 µmol/L was choose to perform subsequent experiments. During 11 days of storage, the BPC, MPV and PDW were not changed significantly between the experimental group and control group, although there was decreasing trend in the BPC and increasing trends in MPV and PDW in the two groups. The rate of Plt PS positive was enhanced during the storage period: the rate of PS positive in experimental group increased from 7.69 ± 1.82% to 10.74 ± 1.78% while it in control group increased from 11.21 ± 2.03% to 15.37 ± 1.95%, with significant differences between the two groups (P < 0.05) on the 9(th) and 11(th) day of storage, respectively. Soluble P-selectin contents in experimental group on the 9(th) and 11(th) day of storage were 30.19 ± 2.03 ng/ml and 34.52 ± 2.64 ng/mL, respectively, while those in control group were 39.18 ± 2.66 ng/ml and 43.23 ± 2.58 ng/ml, respectively, with significant differences between the two groups (P < 0.05). It is concluded that the extended storage of Plt in M-sol treated with low concentration ANA can potentially alleviate Plt storage lesions.

Identification of a novel HLA allele, HLA-B*35:03:07, by sequencing-based typing / 中国实验血液学杂志

This study was purposed to analyze and identify a novel HLA allele in Chinese population. A new HLA-B allele which is closely related to HLA-B*35:03:01 was initially detected by PCR-SSOP, then DNA sequencing was performed to identify the difference between the novel allele and HLA-B*35:03:01 allele. The result showed that the sequence of the new allele was different from all other known sequence. It differs from the closest matching HLA-B*35:03:01 by a single substitution at position 387 C→G in exon 3, no resulting in amino acid change. It is concluded that this allele is a novel one and has been officially named B*35:03:07 by the WHO Nomenclature Committee.

Analysis of HLA-A, B, and DRB1 polymorphism and genetic distance in Han population living in Yantai and Weihai regions of Shandong province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate allelic and haplotypic polymorphisms of human leukocyte antigen(HLA) genes at A, B and DRB1 loci in Yantai and Weihai Han population and analyze the genetic relationship between Yantai, Weihai Han population and other populations.</p><p><b>METHODS</b>A total of 4062 unrelated Han ethnic individual from Yantai and Weihai regions were genotyped by polymerase chain reaction-sequence specific olignucleotide probe(PCR-SSOP) for HLA-A, B and DRB1 loci. Allelic and haplotypic frequencies were estimated by maximum likelihood estimation method using Arlequin 3.5 software. Genetic distances were computed, and phylogenetic tree was constructed using Mega5.0 software.</p><p><b>RESULTS</b>Respectively 18, 33 and 13 alleles were observed at HLA-A, B and DRB1 loci. The most frequent alleles were HLA-A*02(0.2935), HLA-B*15(0.1485) and HLA-DRB1*15(0.1621). And the most common three loci haplotype was A*30-B*13-DRB1*07(0.0649). A*33-B*58, A*66-DRB1*13 and B*08-DRB1*03 showed the strongest linkage disequilibrium. Yantai and Weihai Han population has the shortest genetic distance with Jilin Han population (0.0034).</p><p><b>CONCLUSION</b>The HLA-A, B and DRB1 loci are highly polymorphic in Han population from Yantai and Weihai, and this population has closest relationship with Han population from Jilin province.</p>

HLA-A, B and DRB1 polymorphism at high-resolution in Han population from southern area of Shandong province in China / 中国实验血液学杂志

This study was aimed to analyze the polymorphism of HLA-A, B, DRB1 alleles at high-resolution level in Han population from southern area of Shandong province in China. 688 randomly selected, unrelated and healthy individual from southern area of Shandong province were genotyped for HLA-A, -B and HLA-DRB1 loci by PCR-SBT. Then, allelic and haplotypic distributions of HLA-A, B and DRB1 were estimated by maximum likelihood estimation method using Arlequin 3.0. The results indicated that a total of 31 HLA-A, 63 HLA-B and 39 HLA-DRB1 alleles were identified in Han Population from southern area of Shandong province. Six HLA-A alleles were found with a frequency greater than 0.05 (A*24:02, *30:01, *11:01, *02:01, *33:03 and *02:06), with a cumulative frequency of 0.7223. For HLA-B locus, there were also six alleles which had a frequency higher than 5% (B*1302, *4403, *5101, B*4601, *1501 and *5801), representing 0.4432 of the all alleles in the population. And four HLA-DRB1 alleles were defined as predominant (DRB1*0701, *1501, *0901and *0803), accounting for 0.5453 of the defined alleles. The most common three-loci haplotype was A*30:01-B*13:02-DRB1*07:01 (0.1151) and the most frequent two-loci haplotype were A*30:01-B*13:02 (0.1303), A*30:01-DRB1*07:01 (0.1157) and B*13:02-DRB1*07:01 (0.1307). It is concluded that the allelic and haplotypic diversities of HLA-A, -B and HLA-DRB1 at high-resolution in Han population from southern area of Shandong province in China provide useful information for HLA matching in transplantation and diseases-associated study in this population.

Identification of a novel HLA allele HLA-B*40:96 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a novel human leukocyte antigen (HLA) allele in Chinese and investigate its inheritance in the family.</p><p><b>METHODS</b>Exceptional reaction pattern was detected in HLA-B locus in HLA typing using Luminex DNA polymerase chain reaction with sequence specific oligonucleotide probe hybridization (PCR-SSOP) assay. A confirmatory test for the novel HLA allele was performed by DNA sequencing based typing of the proband's family.</p><p><b>RESULTS</b>The DNA sequence was confirmed to be a novel HLA B allele. There were 7 nucleotides which differed from the closest matching HLA B*40:06:01 at positions 302(G to A), 309(G to C), 311(A to C), 313(C to G), 314(T to C), 317(G to T), and 319(G to C) in exon 2, which resulted in 5 amino acid changes at codon 101 (Ser to Asn), 104 (Asn to Thr), 105 (Leu to Ala), 106 (Arg to Leu), and 107 (Gly to Arg), respectively. Family investigation indicated that the novel allele was transmitted from the proband's father.</p><p><b>CONCLUSION</b>A novel HLA B allele was identified and officially named as HLA-B*40:96 (GenBank accession No. FJ374890) by the WHO Nomenclature Committee for Factors of the HLA System.</p>

Identification of a novel allele HLA-DRB1*1219 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a novel HLA DRB1 allele in a Chinese leukemia family.</p><p><b>METHODS</b>A new HLA-DRB1 allele was initially detected by polymerase chain reaction-sequence specific primer and unusual reaction pattern by Luminex RSSO, then DNA sequencing was performed to identify the sequence of the novel allele.</p><p><b>RESULTS</b>The DNA sequencing revealed the presence of the new allele which differs from the closest matching HLA-DRB1*120201 by a single nucleotide substitution at position (341 C > T in exon 2), resulting in an amino acid change from Ala to Val at coden 85.</p><p><b>CONCLUSION</b>A novel allele was confirmed by DNA sequencing and has been designated HLA-DRB1*1219 by the WHO Nomenclature Committee.</p>