The Significance of the Nucleic Acid Test (NAT) to Prevent Viral Contamination in Musculoskeletal Tissue Transplantation / A importância da realização do teste de ácidos nucleicos (NAT) para a prevenção de contaminação viral nos transplantes de tecidos musculoesqueléticos

Abstract Objective The present study aims to highlight the significance of the nucleic acid test (NAT) for musculoskeletal tissue donation and to compare the sensitivity of this test on the different available platforms. Method The present study is a retrospective survey in a human tissue bank database and an integrative literature review encompassing the last 10 years. The PubMed portal and the SCOPUS, CINAHL, and Web of Science databases were queried for articles. Results We found no specific studies on the use and sensitivity of NAT in braindead tissue donors. The information presented in the present study consists of specific contents intended for the Brazilian Blood Transfusion Network (Hemorrede Transfusional Nacional, in Portuguese) and internal retrospective data from a tissue bank located at a city in the state of São Paulo, Brazil. Conclusions The NAT is effective in blood samples from living patients. However, since biochemical reactions in braindead patients can be different, specific research, platforms, or both are crucial to tissue banks.

Resumo Objetivo Evidenciar a importância da realização do teste de ácido nucleico (NAT, na sigla em inglês) para doação de tecidos musculoesqueléticos, assim como comparar a sensibilidade deste exame nas diferentes plataformas existentes no mercado. Método Trata-se de um levantamento retrospectivo no banco de dados de um determinado Banco de Tecidos Humanos e de uma revisão integrativa da literatura, operacionalizada nos últimos 10 anos. As buscas de artigos ocorreram no portal PubMed e nas bases de dados SCOPUS, CINAHL e Web of Science. Resultados Não foram encontrados estudos específicos sobre a utilização e a sensibilidade do exame NAT em pacientes doadores de tecidos com morte encefálica (ME), sendo as informações apresentadas no presente estudo conteúdos específicos destinados à Hemorrede Transfusional Nacional e aos dados retrospectivos internos de um Banco de Tecidos do interior do estado de São Paulo, Brasil. Conclusões O exame NAT se apresenta efetivo em amostras de sangue de pacientes vivos. Porém, reações bioquímicas em pacientes com condições de ME podem se apresentar de formas diferenciadas, tornando-se indispensáveis a realização de pesquisas específicas e/ou a indicação de plataformas aos Bancos de Tecidos.

Characteristics of viral shedding in people infected with SARS-CoV-2 during difference stages / 中国热带医学

@#Abstract: Viral shedding of SARS-CoV-2 is a continuous dynamic process, which can be divided into latent stage, initial stage, peak stage and decreasing stage according to the characteristics of viral shedding. After being infected with SARS-CoV-2, the infected person generally stays in the latent period for 1-3 days, which is characterized by continuous negative nucleic acid test results and no infectiousness, and the risk of infection for close contacts is very low. At the initial stage of viral shedding is characterized by a rapid decline in the Ct value of nucleic acid tests in a short time, and clinical symptoms gradually appear. The infectiousness of the infected person gradually increases during this period, and the risk of infection for close contacts also gradually increases, but it is still in the early stage of infection, the possibility of viral shedding is low, and the risk of infection of secondary close contacts is low. The peak of viral shedding is characterized by low Ct value in nucleic acid test and obvious clinical symptoms; during this period, the infected person is the most infectious, and the risk of infection of the contact is the highest, so the scope of close contacts should be expanded appropriately. The decreasing period is characterized by the gradual increase of Ct value of nucleic acid test and the gradual disappearance of clinical symptoms; during this period, the infectiousness of the infected person gradually decreases to disappear. In an outbreak, an infected person in the decreasing phase is more likely to be an early infected person in the transmission chain. If infected individuals in the decreasing phase are found in an area without a SARS-CoV-2 epidemic, it suggests that the local outbreak epidemic has been spreading for some time and may be larger in scale. According to the characteristics of viral shedding, risk personnel can be determined more scientifically and accurately, so as to minimize the risk and reduce the waste of epidemic prevention resources.

Expert consensus on organ donation from patients infected with novel coronavirus in China / 器官移植

The spread, prevention and control of novel coronavirus infection and the potential risks and uncertainties of novel coronavirus transmission from donor to recipient have brought serious impacts and great challenges to organ donation and transplantation. There is increasing evidence that the use of non-pulmonary organs (kidney, liver and heart) from novel coronavirus infected donors carries a low risk of transmission, regardless of whether they were symptomatic at the time of acquisition. Delaying organ donation after the death of those who are positive for novel coronavirus antigen or nucleic acid testing, and then waiting until turns negative, will result in the discarding of a significant number of organs that are medically suitable for transplantation. In order to maximally meet the demand for transplantation in patients with end-stage organ failure, Branch of Organ Transplantation of Chinese Medical Association organized relevant experts formulated the "Expert consensus on organ donation from patients infected with novel coronavirus in China" after citizen' s death by taking into account the epidemic situation of novel coronavirus infection in China and the clinical practice of organ donation and transplantation, and by referring to relevant research results and clinical research evidence at home and abroad. It aims to provide recommendations and references for the procurement and application of donor organs from patients infected with novel coronavirus.

Diagnosis and treatment strategies for solid organ transplant recipients with SARS-CoV-2 infection / 器官移植

Due to long-term use of immunosuppressive agents, solid organ transplant recipients (SOTR) belong to high-risk populations of multiple pathogenic infection, including SARS-CoV-2. In addition, SOTR are constantly complicated by chronic diseases, such as hypertension and diabetes mellitus, etc. After infected with SARS-CoV-2, the critically ill rate and fatality of SOTR are higher than those of the general population, which captivates widespread attention from experts in the field of organ transplantation. Omicrone variant is currently the significant pandemic strain worldwide, rapidly spreading to more than 100 countries worldwide and causing broad concern. According to the latest international guidelines on the diagnosis and treatment of SARS-CoV-2 infection and relevant expert consensus in China combined with current domestic situation of SARS-CoV-2 pandemic and China's "diagnosis and treatment regimen for SARS-CoV-2 infection (Trial Version 10)", the epidemiology, clinical manifestations and prognosis, diagnosis, clinical classification and treatment of SARS-CoV-2 infection were briefly reviewed.

Analysis on the influencing factors for re-positive nucleic acid test result in discharged COVID-19 patients in Chengdu / 中国胸心血管外科临床杂志

@#Objective    To analyze the influencing factors for re-positive nucleic acid test in discharged corona-virus disease 2019 (COVID-19) patients in Chengdu, Sichuan Province, and to provide data support for the epidemics prevention and control. Methods    The clinical data of 660 discharged COVID-19 patients from January 23, 2020 to February 28, 2021 in our center were retrospectively analyzed. The patients were divided into two groups according to the reexamination of virus nucleic acid, including a negative group [549 patients, including 428 males and 121 females with a median age of 33.0 (28.0, 48.0) years] and a positive group [111 patients, including 76 males and 35 females with a median age of 39.0 (28.0, 51.0) years]. The clinical data of the two groups were compared. Results     The re-positive rate of the discharged patients was 16.82%. Univariate analysis showed that the re-positive rate of females was higher than that of males (χ2=4.608, P=0.032). The re-positive rate of confirmed patients was higher than that of asymptomatic infected patients (χ2=8.140, P=0.004). The re-positive rate of domestic patients was higher than that of imported patients (χ2=9.178, P=0.002). The counts of CD3+ (P=0.038), CD4+ (P=0.048) and CD8+ (P=0.040) T lymphocytes in the negative group were higher than those in the positive group. The binary logistic regression analysis showed that the clinical classification and CD8+ T lymphocyte count were independent risk factors affecting the recurrence of virility. Conclusion    The gender, origin, T lymphocyte subsets count and clinical type are the influencing factors for re-positive result, and clinical type and CD8+ T lymphocyte count are the independent influencing factors for re-positive result. Therefore, improving the immunity of infected patients, as well as early detection and timely treatment are effective means to reduce the re-positive occurrence.

Performance verification of domestic nucleic acid testing system in blood stations based on T/CSBT / 中国输血杂志

【Objective】 To explore the performance verification of the Shengxiang automatic NAT system for HBV DNA, HCV RNA and HIV RNA-1 using PCR-fluorescence in the laboratories of blood stations, in order to meet the requirements of T/CSBT and ensure the quality of nucleic acid detection. 【Methods】 Samples used in the external quality assessment (EQA) of National Center for Clinical Laboratories of the year 2022 were taken to verify the concordance. The standard materials of HBV DNA, HCV RNA and HIV RNA-1 were used to verify the analytical sensitivity, endogenous interfering substances, repeatability, anti-cross contamination ability and stability. 【Results】 The concordance rate of 20 EQA samples was 100%. The analytical sensitivity of HBV DNA, HCV RNA and HIV RNA-1 were all reactive and met T/CSBT. The yielding of HBV DNA, HCV RNA and HIV RNA-1 was affected little with lipemia at 3g/L and hemolysis at 4g/L. The coefficients of variation(CV) of intra-assay and inter-assay which met T/CSBT were all less than 5%, and the intra-assay variation coefficient was less than the inter-assay variation coefficient. The test results of 40 negative samples tested for cross contamination resistance were 100% negative, and 40 positive samples of HBV with 10 000 IU/mL were 100% positive. The stability verification results showed that the detection rate of weak positive samples was 100%. The coefficient of variation of the test results of the reagent after 1 and 5 freeze-thaw cycles were less than 5%,and the difference between the detection Ct value of reagent underwent once freeze-thaw and five-time freeze-thaw was not statistically significant. 【Conclusion】 The analytical sensitivity,endogenous interfering substances, repeatability,anti-cross contamination ability,stability and the compliance rate of domestic Shengxiang Gene automatic NAT system and supporting reagents by PCR-fluorescence method all meet T/CSBT, so it can be used for nucleic acid detection in blood screening in blood station laboratory.

Following up and re-entry of HBV reactive blood donors in Yantai area / 中国输血杂志

【Objective】 To analyze the preliminary screening and follow-up testing data of HBV in Yantai area, and discuss the rationality of following up and re-entry program of HBV reactive blood donors. 【Methods】 Donors who were single reagent reactive by enzyme-linked immunosorbent assay (ELISA) in initial screening but non-reactive by nucleic acid testing (NAT) were followed up. Individual NAT(ID-NAT) was performed for HBV DNA, ELISA for HBsAg, HBsAb, HBeAb, HBeAg and HBcAb, and ECLIA for the detection of HBsAg. 【Results】 A total of 547 blood donors were HBsAg ELISA-/NAT+, and 97 were followed up, among which 24 met the requirements of re-entry while 73 did not. Of the 24 blood donors who met the re-entry requirements, 13 donated blood again, with test results all qualified. 【Conclusion】 The combination of ELISA, ID-NAT, and ECLIA methods for following up detection for HBsAg ELISA+ blood donors is recommended. Blood donors with HbsAb S/CO ≥ 10 and negative results for other tests met the re-entry requirements, with a re-entry rate at 24.74%, and the re-donation qualified rate of blood donors after re-entry was 100%.

HPV B19, HAV and HEV infection among voluntary blood donors in Nantong / 中国输血杂志

【Objective】 To evaluate the viability of nucleic acid test(NAT) of human parvovirus B19 (HPV B19), hepatitis A virus (HAV) and hepatitis E virus (HEV) along with routine NAT for voluntary blood donors, and to analyze HPV B19, HAV and HEV prevalence in Nantong, so as to provide reference for rational blood screening programs. 【Methods】 HPV B19 DNA, HAV RNA and HEV RNA of blood donors in Nantong from November 2021 to May 2022 were detected using NAT, and serological antibody testing was performed on NAT reactive samples. 【Results】 Three HPV B19 DNA was yielded out of 3 440 blood donors, with a positive rate of 0.09%, among which 2 were negative for HPV B19-IgM and 1 was undetermined due to insufficient sample size. HAV RNA and HEV RNA were not detected in 3 440 blood donors. HPV B19, HAV, and HEV NAT were conducted simultaneously with routine HBV, HCV and HIV screening, prolonging the test reports by 20 minutes. 【Conclusion】 Although the HPV B19 DNA, HAV RNA and HEV RNA prevalence among voluntary blood donors in Nantong is low, the risk of transfusion transmitted infection still exsits and can be reduced by NAT.

Analysis of the types and causes of invalid results in nucleic acid test for blood screening / 中国输血杂志

【Objective】 To analyze the reasons for the invalidity of blood nucleic acid test results, and to explore the countermeasures to reduce the invalidity of the test. 【Methods】 From 2019 to 2021, the number of tests performed in our laboratory for Cobas s201 blood nucleic acid screening system and the number of batches and tests with invalid results were counted, and the types and reasons of invalid results were analyzed. 【Results】 From 2019 to 2021, the Cobas s201 nucleic acid detection system detected a total of 5, 420 batches and 127, 950 pools, and the invalid rate of batches and pools were 1.83% and 1.97%, respectively. The types of invalid results can be summarized as improper operation, sample quality problems, invalid quality control (IQC), equipment failure and others. Among them, IQC and equipment failure were the main reasons for invalid results, accounting for 44.51% and 39.96%, respectively. IQC was mainly related to cross-contamination of samples and insufficient mixing of quality control products. Equipment failures mostly occurred in the robotic arm gripper of the nucleic acid extraction instrument and the TC module of the amplification instrument. 【Conclusion】 The laboratory should conduct quality monitoring for invalid results, and take targeted improvement measures, especially to reduce invalid results caused by invalid quality control and instrument failure.

Cost-benefit analysis of hepatitis B virus serological and nucleic acid testing in blood donors / 中国输血杂志

【Objective】 In an effort to prevent transfusion-transmitted hepatitis B infection, universal HBsAg screening, HBsAg+ MP nucleic acid test(NAT) for HBV and HBsAg + individual(ID) NAT were analyzed for cost-effectiveness. 【Methods】 On the basis of screening data and the documented parameter, the number of window period infections, chronic infections and occult infections was constructed, and cost-benefit analysis was conducted. 【Results】 Of 132 208 donations, the yield rate of ID NAT for HBsAg-/DNA+ (0.11%) was significantly higher than HBsAg+ MP NAT(0.058%). Furthermore, the predicted preventing transfusion transmitted HBV cases by ID NAT is 1.25 times as that by MP-6 NAT, so did the benefits. The cost-benefit of the three screening models were 1∶63.6、1∶28.6 and 1∶53.4. 【Conclusion】 Universal HBsAg in combination with ID HBV NAT screening was the most effective among all screening strategy. It is necessary to applied HBsAg and ID HBV NAT screening for the safety of blood transfusion.

Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay / 南方医科大学学报

OBJECTIVE@#To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.@*METHODS@#We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.@*RESULTS@#This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.@*CONCLUSION@#By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.

One-step multiplex nested real-time RT-PCR assay for 2019-nCoV and Influenza A/B viruses detection / 中华检验医学杂志

Objective:To develop a single-tube one-step multiplex nested real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the simultaneous detection of 2019-nCoV, influenza A virus, influenza B virus and internal-control with human-derived gene.Methods:This study included 30 positive specimens for 2019-nCoV nucleic acid detection and 336 screening specimens collected from the arrivals at Guangzhou Baiyun Airport between February 2020 and February 2022. Sixty-four positive specimens of other respiratory pathogens were also collected from the arrivals at Guangzhou Baiyun Airport during the three-year period before the occurrence of COVID19 outbreak in 2020, and 7 positive viral strains of respiratory pathogens were provided by collaborative laboratories. In order to establish a set of multiplex nested real-time RT-PCR assay, a group of primers and probe combinations for a multiplex nested real-time RT-PCR was designed and screened according to a selection of nucleotide conserved regions of the ORF and N genes of 2019-nCoV and the M gene of influenza A and B viruses, while nested amplification primers and probe for the internal-control with human-derived gene were introduced. Then the prepared pseudovirus-positive quality control and sample discs were applied to evaluate the sensitivity and specificity. Clinical specimens were performed to validate the applicability of the method.Results:The results show that the established one-step multiplex nested real-time RT-PCR assay can specifically detect 2019-nCoV and influenza A and B viruses, with the limit-of-detection of about 125 copies/ml for 2019-nCoV and about 250 copies/ml for influenza A and B viruses. Totally 101 positive samples of various respiratory pathogens were detected, showing that the detection sensitivities of 2019-nCoV and influenza A and B viruses were 96.67%, 92.86% and 96.15%, respectively, with the specificity of 100%. No false-positive detection was found in the applied detection of more than 300 clinical samples.Conclusions:A one-step multiplex nested real-time RT-PCR assay for 2019-nCoV, influenza A and B viruses and human-derived gene internal-control was developed. The assay has good sensitivity and specificity and can be used for rapid screening of 2019-nCoV and influenza A and B viruses in high-volume samples.

Laboratory testing techniques for monkeypox virus / 中华皮肤科杂志

Monkeypox is a zoonotic viral disease caused by monkeypox virus infection. Monkeypox has become a public health emergency of international concern, since it first spread widely in many regions outside Africa in 2022. Accurate and effective detection methods are particularly important for the diagnosis and screening of monkeypox virus infection. This review summarizes laboratory testing techniques for monkeypox virus in recent years, and compares principles and detection performance of microscopy, culture, nucleic acid testing and immunological methods.

Discriminatory positive rate of individual donor-nucleic acid test mode of blood station laboratories in the Beijing-Tianjin-Hebei Region / 中国输血杂志

【Objective】 To analyze the discriminatory positive rate(DPR)of individual donor-nucleic acid test (ID-NAT)mode of blood screening laboratories in the Beijing-Tianjin-Hebei Region, explore the possible reasons for DPR differences among blood station laboratories and the measures to lesson the differences, in order to lay a foundation for realizing the homogenization of detection quality of blood screening laboratories in Beijing-Tianjin-Hebei Region. 【Methods】 The number of triplex-positive samples and discriminatory -positive samples of A, B, C, and D blood station laboratories, which submitted to ID-NAT system, in Beijing-Tianjin-Hebei Region from January to December 2018 were collected by a questionnaire of Quality Supervise Index of Blood Station Laboratories in Beijing-Tianjin-Hebei Region. The triplex-positive samples were divided into solo-positive samples(NAT+ ELISA-) and dual-positive samples(NAT+ ELISA+ ). The changes of total DPR of A, B and C blood screening laboratories in different months was analyzed and compared respectively. The differences of total DPR of ID-NAT, DPR of NAT+ ELISA+ samples, and DPR between NAT+ ELISA-samples and NAT+ ELISA+ samples of A, B, and C blood screening laboratories during January 2018 to December 2018 was analyzed and compared. The difference of DPR of NAT+ ELISA-samples among A, B, C, and D blood station laboratories was also compared. 【Results】 Significant difference in total DPR was noticed in different months of A, B, and C blood station laboratories from January to December 2018(P<0.05), with the highest DPRs of A, B and C laboratory at 91.67%, 72.73%. and 80.39%, the lowest DPRs at 65.88%, 21.05%, and 7.69%, respectively. Significant statistical differences in the total DPR and the DPR of NAT+ ELISA+ samples were found among A, B, and C blood station laboratories(P<0.05). Significant statistical differences in the DPR of NAT+ ELISA- samples were found among A, B, C, and D laboratories(P<0.05). The DPR of NAT+ ELISA+ samples of A and B blood station laboratories (95.97% and 85.25%) were significantly higher than those of NAT+ ELISA-samples (36.36% and 30.71%)(P<0.05). However, the DPR of NAT+ ELISA+ samples of C blood station laboratory (32.63%) was significantly lower than that of NAT+ ELISA-samples (44.39%)(P <0.05). 【Conclusion】 There were significant differences in the total DPR, the DPR of NAT+ ELISA-samples and NAT+ ELISA+ samples that were detected by ID-NAT system in 2018 among blood station laboratories in the Beijing-Tianjin-Hebei Region, and the total discriminatory positive rate in different months was also different for the same blood station. It is necessary to explore the reasons leading to the differences and seek solutions in order to achieve the homogenization of detection quality of blood screening laboratories in Beijing-Tianjin-Hebei Region.

Centralized blood screening results in Ningxia / 中国输血杂志

【Objective】 To retrospectively analyze the quality status and annual trend of provincial-level centralized blood screening across Ningxia, and discuss the effect and advantages of the application of centralized blood screening. 【Methods】 The centralized detection, with the combination of enzyme linked immunosorbent assay (ELISA) and nucleic acid test(NAT), in Ningxia from 2016 to 2020 was statistically analyzed, and the sample size, overall unqualified rate and unqualified rate of each item were compared among different regions. 【Results】 There were about 70 000 samples in Ningxia annually, 65% were in Yinchuan city, and 35% in Shizuishan, Wuzhong, Guyuan and Zhongwei city. The unqualified rates of blood screening in above five cities were 1.09% (2 438/223 852), 1.48% (401/27 024), 1.50% (425/28 364), 1.01% (351/34 772) and 1.45% (435/30 002) respectively. Significant differences were noticed in the unqualified rates of HBsAg, anti-HCV, anti-TP, HIV Ab/Ag, ALT and HBV DNA (P0.05) among the five cities. 【Conclusion】 Centralized blood screening at provincial-level in Ningxia can optimize the allocation of laboratory resources and better ensure blood safety, which is of great significance to the construction of urban public health system in Ningxia.

Performance verification of nucleic acid testing project based on ISO 15189 / 中国输血杂志

【Objective】 To explore the performance verification of NAT and its procedures for HBV DNA, HCV RNA and HIV RNA-1 using PCR-fluorescence via Cobas s201 automatic NAT system and supporting MPX V2.0 reagents that applied in the laboratories of blood stations, in order to satisfy ISO 15189 accreditation requirements and ensure the accuracy of NAT results. 【Methods】 Samples used in external quality assessment(EQA) of year 2020 were taken to verify the concordance, Performance evaluation panel and sensitivity verification panel of Roche second-generation NAT system were used to verify the sensitivity/ specificity and the lower limit of detection, respectively.And HBV DNA, HCV RNA and HIV RNA-1 quality control products were used to verify the anti-interference ability. 【Results】 The concordance rate of 40 EQA, samples was 100%. The sensitivity and specificity of Cobas s201 automatic NAT system and supporting MPX V2.0 reagents in detecting HBV DNA, HCV RNA and HIV RNA-1 were all 100%. The lower detection limit for HBV DNA, HCV RNA and HIV RNA-1 all met the requirements of reagent instructions. The yielding of HBV DNA, HCV RNA and HIV RNA-1 were affected little with hemolysis at 500 mg/dL but interfered seriously as lipemia reached 3 300 mg/dL. 【Conclusion】 The concordance rate, sensitivity, specificity and lower detection limit of the Cobas s201 fully-automatic NAT system and MPX V2.0 reagents by PCR-fluorescence method all met the requirements of reagent instructions. The verification of anti-interference ability demonstrated the requirements of ISO 15189 and the needs of blood station laboratories could be satisfied, and the detection methods and procedures can ensure the accuracy of NAT results.

Performance verification of a individual donation nucleic acid detection system for blood screening / 中国输血杂志

【Objective】 To verify the detection performance of a newly introduced individual donation(ID) nucleic acid detection(NAT) system, and to confirm whether its main test parameters meet the expected requirements of blood screening. 【Methods】 Standard serum and plasma negative for HBV DNA, HCV RNA, and HIV RNA were diluted to different multiples samples (0.5~3 times) of the detection system′s limit of detection (LoD). These samples were conducted NAT test for HBV DNA, HCV RNA and HIV-1 RNA, to verify the testing sensitivity, stability, accuracy and anti-interference ability. 【Results】 The sensitivity of 3× LoD concentration of HBV DNA, HCV RNA and HIV-1 RNA was tested by the system, and the yielding rates were all 100%, with 1 ×LoD at 95.0%~100% and 0.5×LoD at 70.0%~90.0%. The intra-assay and inter-assay precision variation coefficient was 2.07%~2.62% and 2.33%~2.88%, respectively. The accuracy of 10 external quality assessment samples of National Center for Clinical Laboratories was 100%. Severe hemolysis and fatty blood had no effect on the detection of samples with 3×LoD concentration of HBV DNA, HCV RNA and HIV-1 RNA. Any combinations by samples with 2 × LoD concentration of HBV DNA, HCV RNA, and HIV-1 RNA were not inhibited by high concentrations of other viruses. Among 2 041 sero-negative samples from blood donor, the NAT yield of this system was 1.67% (34/2 041), which was a little bit higher than that of a imported minipool system (1.66%, 33/2 041) (P>0.05). 【Conclusion】 The ID-NAT system can meet the requirements in terms of sensitivity, stability, accuracy and anti-interference ability, and can be used for blood screening of blood donors.

Detection ability of nucleic acid screen laboratory in blood bank based on NCCL external quality assessment data / 中国输血杂志

【Objective】 To evaluate the laboratory's NAT ability by analyzing the feedback reports of nucleic acid test (NAT) results of external quality assessment (EQA) of National Center for Clinic Laboratories (NCCL), so as to improve the laboratory management details and ensure blood safety. 【Methods】 The data of NCCL NAT EQA of blood screening laboratory of Tianjin Blood Center (a total of five occasions from Jan 2019 to Jun 2021) were statistically analyzed. 【Results】 From Jan 2019 to Jun 2021, the laboratory participated in EQA for five times and all the results were qualified. The test results of NAT EQA HIV RNA/HCV RNA/HBV DNA detected by R1, R2 and R4 were consistent with the reference results. R3 showed false positive results (CT value 40.46) in the single donation detection of sample No.1925 in HCV RNA. Unreported data of the laboratory was that in the first EQA in 2021, the R4 showed false positive results (CT value 35.8) in in the single donation detection of sample No.2113 in HIV RNA. 【Conclusion】 The performance of each NAT screening system in our laboratory is relatively stable except occasional false positive results influenced by every factor. Potential problems can be found and continuously improved by assaying EQA reports and the extended experimental results of EQA samples to further improve the detection ability.

HBV infection in voluntary blood donors in Qingdao, China: Serological and viral characterizations / 中国输血杂志

【Objective】 To study and analyze the serological and viral charactereristics of hepatitis B virus(HBV) infection in voluntary blood donors in Qingdao. 【Methods】 315 520 blood samples of voluntary blood donors were screened by ELISA combined with nucleic acid testing (NAT). All HBsAg-/HBV DNA+ samples were subjected to high-precision viral load detection and five serological markers of HBV. The sequence of HBV S gene was detected by PCR direct sequencing, and virus genotypes and amino acid mutations were analyzed. 【Results】 A total of 604(0.20%)HBV ELISA or NAT reactive samples were detected: HBsAg+ /HBV DNA- in 307(0.10%) cases, HBsAg-/HBV DNA+ in 138(0.04%) and HBsAg+ /HBV DNA+ in 157(0.05%). Among the 138 HBsAg-/HBV DNA+ donors, 118(85.5%) carried anti-HBc, and 45 (32.61%) carried sole anti-HBc and 5 (3.62%) carried both HBsAg and anti-HBc. In viral load detection, 64 were quantitatively negative and 74 were quantitatively positive, of which 42 were HBV DNA 20 IU/mL. 13 HBsAg-/HBV DNA+ samples were successfully amplified and sequenced, and 5 were genotype B, presenting a total of 17 amino acid mutations without any deletion or insertion, and 8 were genotype C, presenting a total of 41 amino acid mutations and 2 amino acid deletions. 【Conclusion】 NAT, in combination of ELISA, provides additional safety in detecting potentially infectious HBV during the window period and occult HBV infection (OBI). The viral load was low in OBI infected donors, and anti-HBc+ was the main manifestation.The dominating HBV genotypes are genotype B and C, suggesting HBsAg amino acid mutations may be related to the formation of OBI.

NAT results of HBV and HCV under different vacuum collection tubes, storage temperature and storage time / 中国输血杂志

【Objective】 To verify the results of HBV DNA and HCV RNA screening under different brands of vacuum collection tubes for blood samples, storage temperature and storage time. 【Methods】 Experiment 1 was conducted as follows: blood samples were collected simultaneously from 52 voluntary blood donors using two brands(divided into group A and group B) of vacuum collection tubes for blood samples. The plasma separation of group A and group B were compared, and the effects of storage time on the NAT yield of HBV DNA and HCV RNA were statistically analyzed. Experiment 2 was conducted as follows: the effects of different storage temperature, time and tubes on the NAT yield of HBV DNA and HCV RNA samples with low viral load in group A and B were verified and compared in the simulated phlebotomy condition. 【Results】 In Experiment 1: After centrifugation, blood plasma layer and cells layer were separated completely in group A(100%, 52/52), but one sample was not well separated in group B(1/52, 1.92%). After 4 to 10 h after collection, blood samples of two groups were centrifuged and screened for HBV DNA, HCV RNA within 24 h. No positive samples were yielded and the Ct values of internal control(IC-DNA and IC-RNA) were uniform. In Experiment 2: Whole blood samples, stored for either 4 h or 6~10 h at 4 ℃ or 25℃ before centrifugation, showed no difference on the NAT-yield of HBV DNA nor HCV RNA samples with low viral load(P>0.05). Ct values of HBV DNA and HCV RNA of group A was similar to those of group B as centrifuged samples were stored for 24 h or 72~104 h at 4℃(P>0.05), but all increased as the storage time prolonged. Ct values of HBV DNA in group A increased from 33.45±0.29(24 h) to 33.82±0.08(72~104 h) and HCV RNA from 35.21±0.20 to 36.12±0.43; HBV DNA from 33.46±0.25 to 34.30±0.60 and HCV RNA from 35.47±0.24 to 36.49±0.51 in group B. 【Conclusion】 Under certain laboratory condition, different storage time, storage temperature and tubes shed few effect on the NAT-yield of HBV DNA and HCV RNA samples with low virus loads. However, it is suggested that the blood sample be detected within 72 h after centrifugation at 4 ℃ storage.