Detection of Synthetic Cannabinoid CUMYL-PEGACLONE in E-Cigarette Oil and Hair / 法医学杂志

OBJECTIVES@#To establish a combined high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) method to detect the synthetic cannabinoid CUMYL-PEGACLONE in e-cigarette oil and hair.@*METHODS@#HPLC-MS/MS and GC-MS were used to establish the detection method of CUMYL-PEGACLONE, and the hair of drug-involved persons and the seized e-cigarette oil were detected.@*RESULTS@#The main mass spectrometry characteristic ions m/z of CUMYL-PEGACLONE measured by GC-MS were 91, 179, 197, 254 and 372. CUMYL-PEGACLONE had a good linear relationship in the mass concentration range of 2-50 ng/mL, and the linear correlation coefficient (r) was greater than 0.99. The limit of detection (LOD) of CUMYL-PEGACLONE in hair was 0.01 ng/mg, and the limit of quantitation (LOQ) was 0.02 ng/mg. The LOD of CUMYL-PEGACLONE in e-cigarette oil was 1 ng/mg, and the LOQ was 2 ng/mg. The average recoveries of CUMYL-PEGACLONE under the attempt at high, intermediate and low levels in blank human hair and e-cigarette oil matrix were 98.2%-132.4% and 93.5%-110.6%, and the intraday and intraday precision were 1.2%-12.9% and 0.7%-2.9%. CUMYL-PEGACLONE was detected in the hair of 15 drug-involved persons. Except for 1 person who was lower than LOQ, the concentration of CUMYL-PEGACLONE in the hair of other 14 persons was 0.035-0.563 ng/mg. The mass fraction of CUMYL-PEGACLONE in 2 e-cigarette oil were 0.17% and 0.21%, respectively.@*CONCLUSIONS@#The established HPLC-MS/MS and GC-MS methods are applied to the detection of HPLC-MS/MS in drug-related cases, which provides strong evidence support for the handling authority to quickly investigate these cases, and also provides a reference for the identification of such substances in future.

Study on HLA nucleotide sequence matching in epitope positions among recipient-donor pairs for allogenic hematopoietic stem-cell transplantation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the human leukocyte antigens(HLA)-A, -B, -Cw, -DRB1 and DQB1 nucleotide sequences between patients waiting for allogenic hematopoietic stem-cell transplantation (HSCT) and donors in Chinese population, and to establish strategy for maximizing optimal donor selection.</p><p><b>METHODS</b>HLA high-resolution typing in a total of 537 recipient-donor pairs was determined by sequence based typing (SBT) method. The nucleotide BLAST tool was used to compare the nucleotide sequences among recipient-donor pairs.</p><p><b>RESULTS</b>Only 16.20% (88/537) of recipient-donor pairs were found to fully match for nucleotide sequences of all HLA-A,-B,-Cw, -DRB1 and -DQB1 loci. Mismatch rate in single locus were 8.38% in HLA-A, 0.74% in HLA-B, 12.29% in HLA-C, 2.42% in HLA-DRB1, and 2.79% in HLA-DQB1, respectively. Mismatch rate in two or multiple HLA loci was 42.65%. Nonpermissive allele mismatch combinations (A 02:01-A 02:06, A 02:06-A 02:07, Cw 03:04-Cw 15:02, Cw 03:03-Cw 04:01, Cw 03:04-Cw 14:02, Cw 03:03-Cw 08:01, DRB1 04:03:01-DRB1 04:05) were detected in single mismatch HLA locus of recipient-donor pairs, mismatches of B 07:05:01-B 07:06, Cw 07:01:01-Cw 07:06 combinations outside of epitope positions were detected in two recipient-donor pairs.</p><p><b>CONCLUSION</b>Our data suggested that attention should be paid in comparing nucleotide sequences between recipient and donor, and in distinguishing nucleotide sequence mismatches within and outside of the epitope positions. These results could serve as guidelines for donor selection.</p>

Analysis of full intronic sequences of HLA-A alleles / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the full intronic sequences of human leukocyte antigen (HLA)-A alleles in Han Chinese.</p><p><b>METHODS</b>The full-length HLA-A alleles, including 8 exons and 7 introns, were amplified with a long-template PCR system from 165 donors from the Chinese Marrow Donor Program (CMDP). The products were cloned into a PGEM-T Vector System and sequenced from both directions. Genetic analysis was performed using a MEGA4.0 software. All sequences were aligned with a ClustalW algorithm. Phylogenetic trees were constructed with a neighbor-joining method. Genetic distances were estimated based on p-distance, and a bootstrap analysis was applied for assessing the confidence limits of the trees.</p><p><b>RESULTS</b>A total of thirty-three full-length sequences of HLA-A alleles, containing 2902-2918 nucleotides, were derived. A total of 138 point mutations and 9 insertions or deletions were found among the 7 introns, which showed remarkable group specificity. Intron 1 appeared to be most polymorphic with the highest average GC content and evolutionary distances. Eight phylogenetic trees were constructed respectively with the derived full-length sequences as well as each of the 7 introns sequences. Based on full-length sequences, sequences of the HLA-A locus were classified into five groups: group I consisted of A*01/03/11/30; group II consisted of A*23/24(A9); group III consisted of A*02/68/69(A2/28); group IV consisted of A*26/34(A10); and group V consisted of A*29/31/32/33/74(A19). The five groups were derived from two ancient lineages, one including groups I and II, and another including groups III, IV and V. No substantial difference was detected between the trees constructed with the 7 intronic sequences, except that group II belonged to different lineages based on introns 2-5 and introns 1 and 7. The A*30 variant cluster was close to group I (A*01/03/11/30) and differed from group V (A19).</p><p><b>CONCLUSION</b>The full-length sequences of 18 alleles have been submitted to GenBank and accepted by the international ImMunoGeneTics database (IMGT). Polymorphisms identified within the introns of HLA-A alleles showed remarkable group specificity. Such sequences seem to have substantially contributed to the recombination of the HLA-A alleles. The A*30 may represent an atypical group in which the rates of gene conversion and mutation have been unusually high.</p>

DRB1 * 1454: the common allele of human leukocyte antigen-DRB1 * 14 in Chinese Han populations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate and compare the distribution of HLA-DRB1 * 14 alleles between the southern and northern Chinese Han populations.</p><p><b>METHODS</b>Human leukocyte antigen (HLA)-DRB1 alleles of 436 southern and 713 northern Chinese Han bone marrow volunteers were genotyped by polymerase chain reaction (PCR)-sequence-based-typing (SBT) method, among them the DRB1 * 1401/1439/1454 ambiguous allele pairs were identified using DRB1 * 14 high-resolution PCR-sequence specific primer (SSP) kits. Also, the clinic samples previously reported as DRB1 * 1401 were re-genotyped using the same PCR-SSP kits. The allelic distribution of DRB1 * 14 in southern and northern Chinese Han population were compared by chi-square test method.</p><p><b>RESULTS</b>Eighty-one ambiguous allele pairs of DRB1 * 1401/1439/1454 and 54 clinic samples previously reported as DRB1 * 1401 were all identified as DRB1 * 1454. Among the 436 Southern Han donors, six subtypes of DRB1 * 14 allele were observed including DRB1 * 1454 (69.57%), DRB1 * 1402 (1.45%), DRB1* 1403 (1.45%), DRB1 * 1404 (4.35%), DRB1 * 1405 (20.29%) and DRB1 * 1407 (2.90%). In the 713 northern Han donors, a total of seven subtypes were observed including DRB1 * 1454 (35.48%), DRB1 * 1403 (12.90%), DRB1 * 1404 (6.45%), DRB1 * 1405 (37.63%), DRB1 * 1407 (4.30%), DRB1 * 1411 (1.08%) and DRB1 * 1412 (2.15%).</p><p><b>CONCLUSION</b>DRB1 * 1454 and DRB1 * 1405 were the most common alleles of HLA-DRB1 * 14 in Chinese Han populations. The distribution of HLA-DRB1 * 14 differ significantly between the southern and northern Chinese Han population, while DRB1 * 1405 showed similar distribution pattern in the two populations but DRB1 * 1454 had higher frequency in southern than in northern Chinese Han population.</p>

Application value of allele frequencies in direct identification of ambiguous HLA genotypes / 中国实验血液学杂志

This study was aimed to investigate the application value of allele frequencies in direct identification of the ambiguous HLA genotypes. The HLA-A, HLA-B and HLADRB1 loci in 658 Chinese Han donor were detected by PCR-SBT method, the ambiguous genotyping samples were identified by using high resolution PCR-SSP and heterozygous ambiguity resolution primers (HAPRs) methods. The relative probability of true genotypes was calculated by using allele frequencies and was compared with true results. The results indicated that the relative probability of true genotype > 95% in 220 HLA-A ambiguous samples, 238 HLA-B ambiguous samples and 107 HLA-DRB1 ambiguous samples were 99.5% (221/222), 95.8% (228/238) and 97.7% (104/107) respectively. As compared with phenotyping results detected by PCR-SSP and HARP methods, the matching ratios for HLA-A, HLA-B and HLA-DRB1 loci were 100% (222/222), 99.6% (237/238) and 99.1% (106/107) respectively, while the mismatch genotypes were observed only in B*3501/5501 and DRB1*1241/1504, the relative probability of them were 40.3% and 2.1% respectively. It is concluded that the detection method using allele frequencies to directly identify the ambiguous HLA genotypes in large scale PCR-SBT genotyping of donors not only can give higher accurate and reliable results, but also is a simple, rapid and cost-saving method. This method has to be used with great care in the identify-test of patient-donor pair before the transplantation.

Analysis of HLA haplotype frequency and linkage disequilibrium in patients with acute lymphoblastic leukemia from Northern Chinese Han / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the difference between the frequencies of HLA-A-B, B-DRB1 and A-B-DRB1 haplotype, as well as their linkage disequilibrium pattern in patients with acute lymphoblastic leukemia(ALL) and healthy controls from Northern Chinese Han.</p><p><b>METHODS</b>The frequencies of HLA-A-B, B-DRB1, A-B-DR haplotypes and linkage disequilibrium were estimated by Expectation Maximization method based on the genotypes of 643 patients with ALL and 2 0359 unrelated healthy donors, and the statistical significance between the two groups were estimated by chi-square test. Linkage disequilibrium was analyzed with population genetic methods.</p><p><b>RESULTS</b>The most common HLA-A-B, B-DRB1, and A-B-DR haplotypes were A30-B13, A2-B46, A33-B58, B13-DR7, B46-DR9, B52-DR15, B58-DR17, A30-B13-DR7, A33-B58-DR17 and A1-B37-DR10 in both groups. The frequencies of A30-B13, A2-B46, A33-B44, B13-DR7, A30-B13-DR7 and A2-B46-DR9 haplotypes and linkage disequilibrium value were significantly decreased (P<0.05) in the patient group than that in the control group. On the other hand, the frequencies of A2-B52, A31-B61, A24- B8, B60-DR9, B27-DR4, B52-DR14, B44-DR17, B27-DR12 and A11-B27-DR12 haplotypes and linkage disequilibrium value were significantly increased (P<0.05) in the patient group than that in the control group.</p><p><b>CONCLUSION</b>There are some common and positive linkage disequilibrium haplotypes in both the ALL patients and the healthy donors in Northern Chinese Han. Interestingly, some haplotypes and their linkage disequilibrium patterns had significantly different distributions between the two groups. The study provided basic data for the relationship of ALL and HLA haplotype and for finding the HLA-A, B, DR matching donors.</p>

Analysis on haplotypes of five HLA loci in southern Chinese Han population by sequence-based typing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the polymorphism and haplotypes of HLA-A, B, Cw, DRB1 and DQB1 loci in Chinese Han population.</p><p><b>METHODS</b>A total of 186 unrelated healthy individuals from southern China were analyzed by sequence-based typing. Two-, three-, and five-locus haplotypes were estimated using the Expectation Maximization Algorithm. RESULTST: Twenty-eight alleles for the HLA-A locus, 49 HLA-B alleles, 24 HLA-C alleles, 29 HLA-DRB1 alleles and 20 HLA-DQB1 alleles were detected. The A*0207-B*4601(10.81%), A*3303-B*5801(6.14%), B*4601-DRB1*0901(6.22%), B*4001*-DRB1*0901(3.78%), DRB1*090-DQB1*0303 (12.16%) and DRB1*1202-DQB1*0301(8.38%), A*0207-B*4601-Cw*0102 (10.75%), A*3303-B*5801-Cw*0302 (5.14%), A*0207-B*4601-DR*0901(5.07%), A*3303-B*5801-DRB1*0301(2.96%), A*0207-B*4601-Cw*0102-DRB1*0901-DQB1*0303(4.87%) and A*1101-B*1301-Cw*0304-DRB1*1501-DQB1*0601(2.43%) were the most common haplotypes in the southern Chinese Han population.</p><p><b>CONCLUSION</b>The results have shown the characteristics of the five HLA loci haplotype distribution and provided more information in anthropology, disease association studies and transplantation.</p>

Demographic characteristics of patients with leukemia waiting for stem cell transplantation in Chinese Marrow Donor Program during 2000 to 2006 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the distributive characteristics for leukemia and to provide scientific reference for its prevention and intervention.</p><p><b>METHODS</b>Microsoft SQL 2005 databases was used to make a mathematical analysis of 3708 patients with leukemia in Chinese Marrow Donor Program (CMDP) from 2000 to 2006. The distributive characteristics were calculated by sex, age and area of patients with leukemia and then compared by constituent ratio and relative ratio statistics method.</p><p><b>RESULTS</b>A total of 3708 cases of leukemia were registered for waiting donor during the period 2000-2006 in CMDP, the age of patients were from 7 months to 69 years, the median age of diagnosis was 24.5 years, standard deviation was 6.7-years-old; males suffered more than females, and the ratio was 1.95: 1 (2451/1257). There were 1202 patients with acute lymphoblastic leukemia (ALL), 1066 with acute myeloid leukemia (AML), 1435 with chronic myeloid leukemia (CML), 5 with chronic lymphoblastic leukemia (CLL), CML was the most common patients. The distributive of 3708 patients with leukemia peak was from 15 to 30 years age group, 542 patients were at the age of 15 years, 559 patients were at the age group above 20 years, 514 patients were at the age above 25, 522 patients were at the age over 30-years-old. ALL patients were accounted for 49.36% (613/1242), AML patients accounted for 27.78% (245/1242), CML patients accounted for 22.78% (283/1242), CLL patients accounted for 0.08% (1/1242) in the age group of under 20 years (childhood group). All subjects were mainly in childhood patients with leukemia; The distributive of patients with leukemia in 30 areas were different, leukemia patients were not registered in one area, 494 patients were at the highest peak, 101 patients were in the median.</p><p><b>CONCLUSION</b>The majority of leukemia patients for waiting stem cell transplantation were registered among children and the adolescents groups, males were suffered more than the females. For children, the major type of leukemia was ALL, being necessary to pay more attention to the education of health, and the precaution of leukemia. The distributive of patients with leukemia for waiting stem cell transplantation was different in 30 areas, and the peak region of leukemia should be in Jiangsu, Guangdong, Shangdong, and Zhejiang provinces.</p>

Establishment of the method to induce and measure human IL-2 in vitro / 中国实验血液学杂志

This study was aimed to establish the quantitative analysis of hIL-2 in culture supernatant by multifunctional Luminex 100. The lymphocytes were separated from ACD-anticoagulated peripheral blood by density gradient method. The lymphocytes were stimulated with PHA for 48 hours, and frozen at -20 degrees C The relative fluorescence units of standard preparations and samples were detected by multifunctional Luminex 100, and the sample concentrations were calculated by standard curve. The results indicated that the regression equation of standard preparation is Lg (RFU) = 1.547 + 0.867 LgC. ANOVA F = 301.7427, p < 0.05 (nu = 6). The analysis of variance showed F = 301.7427, p < 0.05 (nu = 6). The test of regression coefficient showed t = 17.3707 (nu = 6), p < 0.05. It is concluded that method for induction and measurement of human IL-2 in vitro is established. The standard curve established by this way is statistically significant. There is linear relationship between the concentration of hIL-2 and fluorescence intensity.

Sequence analysis of a novel HLA allele B*5618 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify HLA novel allele in Chinese Han individual.</p><p><b>METHODS</b>An unknown HLA-B allele which was similar to HLA-B*5610 was detected by polymerase chain reaction-sequence specific oligonucleotide probes(PCR-SSOP), PCR-sequence specific primer(PCR-SSP) and heterozygous sequence-based typing (SBT) in a Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. The HLA-B*56 allele was amplified separately by using allele-specific primers and sequencing exons 2-4 in both directions. The differences between the novel B*56 allele and B 5610 were identified.</p><p><b>RESULTS</b>There were 4nt changes from B*5610 in exon 3, at nt379 where C>G (codon 127 CTG>GTG, 127 Leu>Val); nt412 where A>G (codon 138 AAC>GAC, 138 Asn>Asp), nt419 where T>C and nt420 where A>C (codon 140 TTA>TCC, 140 Leu>Ser). The sequence was submitted to Genbank and the accession number was EF016753.</p><p><b>CONCLUSION</b>This allele is a novel HLA-B allele, and has been officially named HLA-B*5618 by the WHO Nomenclature Committee in September 2006.</p>

Identification and sequence analysis of a novel HLA-A * 3018 allele / 中国实验血液学杂志

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).

Polymorphism of HLA-B* 40 gene family in Chinese Han population / 中国实验血液学杂志

To investigate the allele distribution of HLA-B* 40 gene family in Chinese Han population and to study its influence on the selection of clinical transplantation donor, the HLA-B genetypes of 381 individuals randomly selected from Chinese National Marrow Donor Project were identified by PCR-SSO, and then all the HLA-B* 40 positive samples from the above population and the B* 40 homozygote samples received from another 1 270 registered donors were analyzed by PCR-SBT and PCR-SSP at high resolution. The results showed that the population of 381 registered donors was examined at HLA-B locus by using Hardy-Weinberg equilibrium, the gene frequency of HLA-B* 40 was 0.1692. Four different HLA-B* 40 alleles (B* 4001, B* 4002, B* 4003, B* 4006) were identified, and the serological specificity was B60 and B61 respectively. The relative frequency of each allele was 0.1192 for B* 4001, 0.0154 for B* 4002, 0.0038 for B* 4003, 0.0308 for B* 4006. The distribution of B* 40 homozygote revealed a certain regularity at high-resolution, B* 40XX (B* 4001 group), at low-resolution; B* 4001 at high resolution; B* 40XX (B* 4002 group), at low-resolution; B* 4002 or B* 4006 or heterozygote of both at high-resolution. It is concluded that in Chinese Han population, predominant allele in HLA-B* 40 gene family is B* 4001, the high-resolution typing may be recommended to use for the selection of clinical transplantation donor.

Study of HLA polymorphism in the 6965 Han bone marrow registry donors / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze human leukocyte antigen (HLA) polymorphism and search for new alleles in Chinese Han population bone marrow registry donors.</p><p><b>METHODS</b>DNA-based HLA genotyping methods were used including PCR-SSP, BST and molecular cloning.</p><p><b>RESULTS</b>A total of 6965 unrelated donors, 4707 from South China origin and 2258 from north, were typed for HLA-A, B, and DRB1 loci. Seventy-two specificities of HLA alleles were identified. The HLA-A25, A34, A74, B41, B42, B53, B73 and B81 that were rarely reported in previously Chinese population studies were identified in this study. Estimation of gene frequency indicated that the blank gene frequency was less than 0.2% for HLA-A, 0.25% for HLA-B and 0.70% for HLA-DRB1 loci. Three novel alleles were identified and officially assigned by the World Health Organization (WHO) Nomenclature Committee as A*0253N, A*1114 and B*5610.</p><p><b>CONCLUSION</b>Large-scale DNA-based HLA genotyping used in bone marrow registry donors is highly accurate and reliable for estimating gene frequency and searching for new alleles. The discrepancy of HLA gene distribution between South and North China Han population showed the necessity of setting the more regions in South and North China to screen the bone marrow registry donors for bone marrow transplant.</p>

Experimental study on quantitative monitoring engraftment of an adult with mixed umbilical cord blood transplantation / 中国实验血液学杂志

The purpose of this research was to monitor quantitatively and study the dynamic changes and development rules of engraftment, chimera types, as well as relative amount of donor cells after allogeneic transplantation of mixed umbilical-cord blood from two units. An adult patient with acute myeloid leukemia received two units HLA one locus mismatched unrelated umbilical cord blood transplantation (2.5 x 10(7)/kg karyocytes in umbilical cord blood unit 1, and 1.53 x 10(7)/kg karyocytes in umbilical cord blood unit 2). Nine STR loci of the blood sample before and after transplantation were determined by quantitative detecting technique with fluorescence labeling polymerase chain reaction, while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. Then the relative proportion of chimera from two units of umbilical-cord blood in the patient after transplantation was calculated according to the differential gene peak areas of two donors on 377XL DNA sequencer after fluorescence scanning, and the engraftment level and the development rules of donor cells were analyzed. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment. The results showed that two umbilical cord blood units at 15 days after transplantation were engrafted simultaneously and revealed a complete chimerism of the two. The relative amounts of chimera from unit 1 vs that of unit 2 were 51.3% vs 48.7%; subsequently relative amounts of chimera from unit 1 went up to 70.0% at 30 days, and that from unit 2 declined to 30.0%. However, at 52 days, only the genotype of umbilical cord blood unit 1 was detected, so that the engraftment turned to a complete chimerism of a single donor type. The one with fewer karyocytes was rejected and the one with more karyocytes finally engrafted in long-term. It is concluded that quantitatively detecting STR chimera with fluorescence labeling polymerase chain reaction can depict precisely the engraftment level and the change course of two umbilical cord blood units. It provides an accurate and reliable experimental basis for clinical umbilical cord blood application and donor selection, and is proved to be feasible for adult transplantation by using dual unit of umbilical-cord blood with HLA one locus mismatched at the same time.