Blood&Genomics

Lewis (Le) blood group system phenotypes and genotypes

Gradimir Misevic

2019, 3(1): 1-8. doi: 10.46701/APJBG.2019012019109

Abstract(1209) PDF(1471KB)(1130)

Abstract:
The Lewis blood group system in humans, designated with number 007 and symbol Le, consist of two different fucose containing carbohydrate antigen structures abbreviated Le^a+ (LE-1) and Le^b+ (LE-2). The expression of these two carbohydrate sequences are phenotype determinants. The Le^a+ antigen sequence is triasaccharide β-D-Galp-(1- 3)-[α-L-Fucp-(1-4)]-D-GlcpNAc. The Le^b+ antigen sequence is tetrasaccharide α-L-Fucp-(1-2)-β-D-Galp-(1-3)- [α-L-Fucp-(1-4)]-D-GlcNpAc. Biosynthesis of Le blood group glycan antigens is catalyzed by fucosyltransferase 2 (FUT2) and fucosyltransferase 3 (FUT3) enzymes. These enzymes are encoded by two dominant autosomal genes named FUT2, also referred as secretory (Se) gene, and FUT3, both having multiple alleles. These two genes determine Lewis blood group genotypes. Sequencing of fucosyltransferase genes, RNAs and fucosyltransferase enzymes and the determination of their structures, together with functional studies including spatial and temporal expression patterns, showed preservation of the catalytic domain within prokaryotes and eukaryotes, with a high level of diversity in structural and functional properties. Six different Le blood group phenotypes exist, taking in account that Le and ABO blood group antigens both comprise terminal sequences on the same branched glycan molecules: Le^a+b-, Le^a-b+, Le^a-b-, Le^a+b+, ALe^a-b+, and BLe^a-b+. Le antigens are part of glycan structures synthetized in glycolipids or glycoprotein form in the endoderm and not in the erythrocyte precursor cell. They are present on the cell surface of blood cells, in plasma and in secretory fluids. Le glycolipids are adsorbed from plasma on the cell surface of erythrocytes, platelets, lymphocytes and endothelial cells.

Experimental model systems for preclinical research on Waldenström macroglobulinemia

Fumou Sun, Yan Cheng, Siegfried Janz

2019, 3(1): 9-18. doi: 10.46701/APJBG.2019012019114

Abstract(974) PDF(551KB)(1049)

Abstract:
Waldenström macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma of mature IgM⁺ B-lymphocytes that warrants additional research to increase therapeutic options, enhance quality of life, and improve survival of patients with WM. Here we concluded a miniseries of short reviews on the diagnosis and treatment ^[1], natural history ^[2] and putative cell-of-origin of WM ^[3] with a brief survey of preclinical experimental model systems available for fundamental and translational research studies on this enigmatic neoplasm. The model systems comprise of: ① continuous tumor cell lines, three of which are well authenticated and demonstrated to be derived from the patient's index tumor; ② human-in-mouse xenografts that rely on immunodeficient laboratory mice, adapted to carry small fragments of implanted human bone, to provide a suitable microenvironment for incoming lymphoma cells; and ③ genetically engineered mouse models (GEMMs) of neoplastic B-cell development, in which WM-like tumors arise spontaneously in the presence of fully functional innate and adaptive immune systems. Because none of the models developed thus far are perfect, additional efforts are required to achieve a better preclinical representation of disease characteristics of WM. To achieve that goal, the active involvement of basic and clinical research experts from China is called for, so novel drugs and immunotherapies for WM will reach clinics sooner, thereby ensuring the future of patients with WM will be brighter.

The polymorphisms of HLA class Ⅰ and Ⅱalleles were not associated with severe acute respiratory syndrome among recovered patients in Beijing: a case-control study

Dongmei Li, Dongjing Liu, Yi Zha, Tao Wu, Yan Qiu

2019, 3(1): 19-25. doi: 10.46701/APJBG.2019012018140

Abstract(829) PDF(3161KB)(1180)

Abstract:
Severe acute respiratory syndrome (SARS) is a viral respiratory illness caused by a novel coronavirus (SARS-CoV), which emerged as a pandemic in 2003. The mechanism of the immune reaction initiated by SARS-CoV still remains unclear. Here we aimed to describe the genetic patterns of high-resolution HLA-A, -B, -C, -DRB1, and -DQB1, loci in recovered SARS patients from Beijing and examine the association between HLA genes and susceptibility or resistance to SARS. A total of 70 recovered Chinese Han SARS patients were recruited to donate convalescent plasma in 2003. HLA high-resolution typing was carried out using sequence based typing (SBT). Allele frequencies were calculated by direct counting, and were compared with the frequencies of HLA alleles of donors recruited by the China Marrow Donor Program between 2002 and 2015 using Fisher's exact test. Significance of association was defined according to the Bonferroni method for multiple comparisons. We observed 20, 35, 21, 25, and 17 alleles respectively at HLA-A, -B, -C, -DRB1, and -DQB1 loci among the 70 recovered patients. We identified 12 alleles (HLA-A*02:10, -A*02:93, -A*03:02, -B*08:01, -B*15:152, -B*37:01, -DRB1*10:01, -DRB1*11:03, -DRB1*14:10, -DRB1*14:12, -DRB1*15:02, and -DQB1*05:10) showing a nominal association with SARS (P<0.05), but none remained significant after Bonferroni correction. The study suggests that high-resolution HLA alleles are unlikely to contribute significantly to the susceptibility or resistance to SARS-CoV infection in the northern Chinese population.

Establishment of laboratory quality control indicators for NAT on blood donors' samples

Ziyi He, Shaobin Chen, Lin Yu, Qing Wang, Jiaoli Zou

2019, 3(1): 27-33. doi: 10.46701/APJBG.2019012018135

Abstract(864) PDF(3597KB)(1190)

Abstract:
In order to establish and screen nucleic acid test (NAT) quality control indicators for blood donor samples, C_T value of each test item were detected by NAT, internal and external quality controls, the number of unsuccessful mixed samples, the split positive rate, the rate of detection efficiency, and the equipment failure rate were collected. The "instant method" is used to establish the Levey-Jennings quality control chart, the mixed test positive rate, the rate of unqualified sample. Possion distribution is used to establish the quality control chart, and it is easily to operate and monitor NAT effectively. The results displayed that quality control charts were established, including unqualified sample Possion distribution quality control chart, the rate of equipment failure quality control chart, the failure rate of reagent batch and inefficient quality control chart, test result positive of Possion distribution quality control chart, quality control charts of NAT positive Possion distribution probability, the rate of split positive quality control charts, quality control charts of unsuccessful mixed samples, quality control chart of reagents effective rate, internal quality control chart, correctness, and NAT qualitative quality control chart. The study established quality control indicators throughout the whole NAT process, which were able to effectively improve NAT quality control and the efficacy of laboratory management.

Detection of RHD zygosity in China: using Syber Green Ⅰ real-time polymerase chain reaction based on high-resolution melting curve analysis

Minyu Zhou, Zhiyuan Xu, Jingyan Chang, Xiaojie Zhu, Yu-Shiang Lin

2019, 3(1): 35-39. doi: 10.46701/APJBG.2019012019103

Abstract(881) PDF(3067KB)(1071)

Abstract:
Due to relatively higher mutation frequencies in Chinese individuals with the RHD-negative phenotype[25% for 1227 G>A RHD elution and 5% for RHD1-RHCE(2-9)-RHD10], Rhesus box analysis is rarely used in China. Here, quantitative real-time polymerase chain reaction (qPCR) with a high-resolution melting curve mode and a matrix mix containing Syber Green Ⅰ were used to sequence specific primers of 1227 G>A and RHD exons 1, 5, and 10 in two families, consisting of two parents and two children per family (n = 8). The samples with RHD gene dele- tion homozygous/heterozygous, 1227 G>A heterozygous with RHD gene deletion and normal RHD, normal RHD homozygous/heterozygous, and RHD1-RHCE(2-9)-RHD10 homozygous/heterozygous status were all included. All samples were screened using RHD exon genotyping, Sanger sequencing, and Rhesus box analysis. DNA sample quality was maintained at 68~72 ng/μL, and OD_260/280 at 1.7~1.9. The Tm ratio of RHD exon 1 (87 ℃ ) to internal control (77℃ ) was 2.49~2.67 and 2.09~2.35 in subjects with RHD exon 1 homozygous and heterozygous, respectively; the Tm ratio of RHD exon 10 (81℃ ) to internal control (77℃ ) was 5.01~6.11 and 3.34~4.31 in subjects with RHD exon 10 homozygous and heterozygous, respectively; the Tm ratio of RHD exon 5 (83℃ ) to internal control (77℃ ) was 3.98~4.75, 3.02~3.45, and 0.03 in subjects with RHD exon 5 homozygous, heterozygous, and deletion homozygous, respectively; the Tm ratio of 1227A (87℃ ) to internal control (77℃ ) was 1.11, 0.51, and <0.03 in subjects with 1227A heterozygous, 1227A homozygous (exon 9 deletion), and wild type, respectively. The results suggest that using the primers of Tm ratio in comparison with an internal control is an effective way to detect RHD gene deletion or RHD-RHCE hybrid variant allele carrier. The method can also be used to calculate the mother-newborn RHD phenotype proportion and assist pedigree analysis.

Method verification study for nucleic acid test system

Libo Zhang, Chengping Ma, Rongrong Pang, Yuan Ke, Min Huang, Wenping Han, Xiaojun Lu

2019, 3(1): 41-45. doi: 10.46701/APJBG.2019012019111

Abstract(4034) PDF(2405KB)(1681)

Abstract:
To identify a suitable method for nucleic acid test(NAT) system verification, several methods were used to verify the system's key parameters, such as the lowest limit of detection, specificity, accuracy and anti-interference ability. The lowest limit of detection for Grifols' Procleix Tigris System were HBV DNA 3.1 U/mL, HCV RNA 5.0 U/mL, HIV RNA 21.2 U/mL; accuracy 100%; anti-interference lipemia (triglyceride)< 33.23 mmol/L, hemolysis (hemoglobin concentration)<5 g/L. There were no significant differences between the claimed specification of both the Grifols' Procleix Tigris Systems and reagents, which both met with published test requirements. New equipment installation or regular verification are necessary to ensure the reliable operation of equipment, which ensure the quality of analysis and test. A systematic method was practiced in our laboratory, which was able to confirm that commercial NAT reagents meet the rigorous standards of blood screening. This study provides a very useful model for other blood screening laboratories and NAT kits.

Pedigree analysis for the rare RHD DVa allele: serological and molecular studies

Jingjing Gao, Xiongpeng Zhu, Jinzhe Tan, Mingquan Wang, Wenqian Xu, Jhy Sheng Chang

2019, 3(1): 47-50. doi: 10.46701/APJBG.2019012019102

Abstract(883) PDF(619KB)(1124)

Abstract:
Until now, worldwide more than 80 different alleles producing weak D phenotypes have been identified. Here we identified rare RHD DVa alleles in Chinese individuals associated with weak expression of D antigen and an RHD phenotype resembling DVI. Multi-monoclonal anti-D antibodies were used to identify the RHD phenotyping for rare RHD DVa. RHD genotyping was used to confirm the presence of RHD exons and identify RHD, RHCE hybrids and exon deficiencies. Sanger sequencing was used to identify nucleotide polymorphisms in RHD exons. Pedigree analysis demonstrated RHD DVa allele alterations of 667 T>G, 676 G>C, 697 G>C, 712 G>A, 733 G>C, 744 C>T and 1227 G>A, which means the proband's alleles were RHD DVa-3 [also called RHD-CE(5)-D] and 1227 G>A. The results also demonstrated RHD DVa and the original RHD Va allele without 1227 G>A. The study suggests that RHD phenotyping is a superior strategy for the molecular analysis of RHD variant in Chinese subjects, and for understanding related polymorphisms and mutations.

The prevalence of alloantibody in the population of Xinjiang area of China

Limin Zhang, Xiao E, Haiying Wang, Mingjun Huang, Gulinuer Kuerban, Xiaoli Sun, Senru Zhang, Yanchao Xing

2019, 3(1): 51-53. doi: 10.46701/APJBG.2019012018139

Abstract(876) PDF(2559KB)(1053)

Abstract:
Alloantibodies are the major cause of hemolytic transfusion reaction and newborn hemolytic disease. It is highly recommended to screen alloantibodies before transfusion and pregnancy. This report applied the microcolum gel method to screen for available alloantibodies, enrolling 20,098 patients from January 2016 to December 2017 in the Xinjiang General Hospital. Seventy-two patients were found alloantibody-positive, at an overall positive rate of 0.35%. The distribution of alloantibody varied according to age, gender or treatment. The patients aged 70 plus and the patients admitted in the Department of Hematology and Department of Gynecology & Obstetrics had a higher incidence rate of alloantibodies. By using 10 screening cell panel systems, 9 types of alloantibodies including anti-D, anti-E, anti-e, anti-C, anti-c, anti-M, anti-s, anti-Fy^b and anti-Ce were identified. This study suggests that the transfusion of red blood cells(RBCs) and pregnancy are the main causes of alloantibodies.

High CCNB2 expression correlates with poor prognosis in hepatocellular carcinoma

Xiaolu Zhai, Yingjing Wang, Pei Chen, Mengjing Sun2, , Jianwei Qiu, Yao Wang

2019, 3(1): 55-62. doi: 10.46701/APJBG.2019012019110

Abstract(815) PDF(1793KB)(1145)

Abstract:
Cyclin B2 (CCNB2), a member of the cyclin protein family, has a key role in the progression of G2/M transition. However, the clinical value of CCNB2 in hepatocellular carcinoma (HCC) is still unknown. The aim of our study is to identify the role of CCNB2 in HCC patients. Immunohistochemical analysis using tissue microarray (TMA) was employed to evaluate the expression of CCNB2 in HCC and the correlation between CCNB2 expression and clinicopathological features in HCC patients. The relationship between CCNB2 expression and the prognosis of HCC patients was analyzed using Oncomine and Kaplan-Meier Plotter online resources. High CCNB2 cytoplasmic expression was observed in 77.22% of patients with HCC, which was related to differentiation (P<0.001), tumor diameter (P=0.025), and hepatitis B virus infection (P=0.008). High CCNB2 nuclear expression was seen among 43.43% of cases, which was associated with differentiation (P=0.001). CCNB2 levels were inversely proportional to patient prognosis. The study suggests that CCNB2 expression could be an effective prognostic biomarker for HCC.

Role of thromboelastography in clinical emergency trauma patients

Fei Geng, Xiaoxin Liu, Ping Fang, Haiying Hu, Xia Deng, Wenjuan Wang, Yin Li, Youbei Lai, Xiaoxiao Zhong

2019, 3(1): 63-66. doi: 10.46701/APJBG.2019012019108

Abstract(887) PDF(251KB)(1052)

Abstract:
This study aimed to investigate dynamic changes in thromboelastography (TEG) and evaluate its value for emergency trauma patients. TEG values (R, K, Angle, MA and CI) were detected l, 3, and 7 days post trauma. Followup prognosis data were recorded among all patients and correlations between TEG changes and prognosis were assessed. The results revealed that the K and R volumes of the poor prognosis group were obviously higher than those of the good prognosis group at each time point; while the Angle, MA, and CI volumes of the poor prognosis group were relatively lower than those of the good prognosis group, and showed a little rising tendency. This study suggests that variables measured by TEG were closely associated with prognosis evaluation, and highlighted the dangers of coagulation disorder at early stage. TEG is of benefit in evaluating the function of coagulation in clinical emergency trauma patients.

Optimization and performance validation for the fully automatic blood type analysis system

Rongrong Pang, Libo Zhang, Yong Wang, Wenping Han, Min Huang, Qiang Fu

2019, 3(1): 67-72. doi: 10.46701/APJBG.2019012019112

Abstract(793) PDF(325KB)(1056)

Abstract:
To evaluate and validate the application of fully automatic blood type analysis system parameters under actual lab conditions. All key system parameters were optimized and validated accordingly. The optimized parameters were centrifugal speed at 550 rpm; centrifugal time 20 min; resuspension speed 1,200 rpm; resuspension time: 45 s; incubation temperature 25℃ ; incubation time 400 s; incubation rate: 0 rpm. The sampled red blood cell concentration was 3%. The ratio of plasma to red blood cells reagent was 60 μL:30 μL; the ratio of antibody (reagent) to sampled diluted red blood cell was 30 μL:30 μL. After applying our key parameters for optimization and validation, the automatic blood type detection system's performance was found to meet the relevant requirements, effectively improving the accuracy and reliability of the detection system.

2019 Vol. 3, No. 1