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Background Recent studies have successfully implemented next-generation sequencing (NGS) in HLA typing. We performed HLA NGS in a Korean population to estimate HLA-A, -B, -C, and -DRB1 allele and haplotype frequencies up to an 8-digit resolution, which might be useful for an extended application of HLA results. Methods A total of 128 samples collected from healthy unrelated Korean adults, previously subjected to Sanger sequencing for 6-digit HLA analysis, were used. NGS was performed for HLA-A, -B, -C, and -DRB1 using the AllType NGS kit (One Lambda, West Hills, CA, USA), Ion Torrent S5 platform (Thermo Fisher Scientific, Waltham, MA, USA), and Type Steam Visual NGS analysis software (One Lambda). Results Eight HLA alleles showed frequencies of ≥10% in the Korean population, namely, A*24:02:01:01 (19.5%), A*33:03:01 (15.6%), A*02:01:01:01 (14.5%), A*11:01:01:01 (13.3%), B*15:01:01:01 (10.2%), C*01:02:01 (19.9%), C*03:04:01:02 (11.3%), and DRB1*09:01:02 (10.2%). Nine previous 6-digit HLA alleles were further identified as two or more 8-digit HLA alleles. Of these, eight alleles (A*24:02:01, B*35:01:01, B*40:01:02, B*55:02:01, B*58:01:01, C*03:02:02, C*07:02:01, and DRB1*07:01:01) were identified as two 8-digit HLA alleles, and one allele (B*51:01:01) was identified as three 8-digit HLA alleles. The most frequent four-loci haplotype was HLA-A*33:03:01-B*44:03:01:01-C*14:03-DRB1*13:02:01. Conclusions We identified 8-digit HLA-A, -B, -C, and -DRB1 allele and haplotype frequencies in a healthy Korean population using NGS. These new data can be used as a representative Korean data for further disease-related HLA type analysis.

Background : We investigated hepatitis B virus (HBV) infection cases, who were HBsAg negative by radioimmunoassay (RIA) and HBV DNA positive for their clinical characteristics, the S gene mutation of hepatitis B virus (HBV), and usefulness of other HBsAg immunoassay. Methods : Among the patients requested for HBV DNA quantification, 16 patients positive in HBV DNA but negative in HBsAg RIA (BNIBT HBsAg Kit, China) were enrolled. The "a" determinant of HBV S gene was sequenced and clinical characteristics were reviewed. Additional HBsAg assay was performed using Architect HBsAg kit (Abbott laboratories, USA) employing chemiluminescent immunoassay method. Results : Eleven of the 16 patients showed multiple mutations in the "a" determinant. These patients received liver transplantation several years ago and have been treated with hepatitis B immune globulin (HBIG) and antiviral drugs. G145R mutation was found in 8 patients and G145K, D144G, and D144A were also frequently found. Among 9 of the 11 patients tested for HBsAg by Architect HBs-Ag kit, 8 showed positive results. Among 4 of the remaining 5 patients, only 2 showed weak positive results (1 IU/mL) in Architect HBsAg kit. Conclusions : HBV DNA-positive/HBsAg RIA-negative results were mostly observed in the patients treated with HBIG after liver transplantation, in whom HBIG escape mutations were found. Majority of these cases were positive in Architect HBsAg assay, and it is recommended to use other HBsAg immunoassay methods that are more sensitive than RIA in the detection limit as well as in the detection of escape mutant in hospitals performing liver transplantation.

Background : Hepatitis B virus (HBV) detected in Korean patients almost belongs to genotype C, which is subdivided into subgenotype C1 (or Cs) and C2 (or Ce). It was recently reported that the risk of hepatocellular carcinoma is different between subgenotype C1 and C2. Thus, we studied the distribution of subgenotypes of HBV in Korean chronic hepatitis B (CHB) patients. Methods : Specimens of 421 patients, who were diagnosed as CHB and underwent antiviral treatment, were used. After sequence analysis for HBV S gene, subgenotype was identified through phylogenetic analysis. Utilizing the same sequence data, the distribution of serotypes was also investigated. Results : Among 421 patient specimens, genotype C was found in 419 (99.5%) and genotype B in 2 (0.5%). Among the genotype C strains, 417 strains were C2 subgenotype and 2 strains were mixed subgenotypes. However, C2 was evidently found even in the mixed sequences. Serotypes of 419 HBV with genotype C were classified as follows: adr, 385 (91.9%), adw, 22 (5.3%), ayr, 2 (0.4%) and mixed serotype, 10 (2.3%). Serotype of both HBV with genotype B was adw. Conclusions : It was found that HBV detected in Korean CHB patients under treatment almost all belong to the C2 (Ce) genotype.

김명희∙차충환∙안동희∙최성은∙오흥범 울산의대 서울아산병원 진단검사의학과 Background : Hepatitis B virus (HBV) DNA quantification is necessary for starting and monitoring of antiviral therapy in patients with chronic hepatitis B. This study was intended to assess the clinical performance of Abbott RealTime HBV Quantification kit (Abbott Laboratories, USA). Methods : The performance was evaluated in terms of precision, linearity, detection sensitivity, cross-reactivity, and carry-over. A correlation with the Real-Q HBV Quantification kit (BioSewoom Inc., Korea) was also examined using serum samples from 64 patients diagnosed with chronic hepatitis B and underwent lamivudine therapy in Asan Medical Center. We verified the trueness of the system by comparing the outputs with the assigned values of the BBI panel (BBI Diagnostics, USA). Results : Within-run and between-run coefficients of variation (CV) were 3.56-4.71% and 3.034.98%, respectively. Linearity was manifested ranging from 53 to 10 9 copies/mL and the detection sensitivity was verified to be 51 copies/mL. None of hepatitis C virus showed cross-reactivity. No cross-contamination occurred when negative and positive samples were alternatively placed in a row. It showed a good correlation with the Real-Q HBV (r 2 =0.9609) and the test results for the BBI panel were also well agreed to the assigned values (r 2 =0.9933). Conclusions : The performance of Abbott RealTime HBV Quantification kit was excellent; thus, it should be widely used in starting and monitoring of antiviral therapy in Korean patients with chronic hepatitis B. (Korean J Lab Med 2008;28:144-50)

Background : To monitor the performance of histocompatibility testing laboratories, HLA proficiency survey in Korea has been conducted biannually since 1996. In this report, we summarized the results of the surveys performed in recent two years (2005-2006). Methods : A total of four proficiency surveys were performed, in which 59-61 laboratories participated. Each survey included three tests for HLA class I (serology and DNA) and class II (DNA) typing and six tests for HLA crossmatch. Results : The overall concordance of serologic typing was 98.9% (355/359) for HLA-A, 97.5% (350/359) for HLA-B, and 94.7% (337/356) for HLA-C. The antigens assigned correctly by less than 95% of the participating laboratories were A26 (93.8%), B38 (94.2%), Cw3/Cw10 (90.9%), Cw6 (94.4%), and Cw8 (74.3%). The overall concordance rates of DNA typing were 99.6% (533/535) for HLA-A, 99.8% (539/540) for HLA-B, and 100% (392/392) for HLA-C. Correct assignment of HLA-DRB1 and -DQB1 was reported by 99.2% (98.1-100%) and 96.7% (88.9-100%) for the generic level and 100% and 95.8% (75-100%) for the allelic level, respectively. On the average 3.8% (0-7.7%) of the total laboratories showed unacceptable results in the crossmatch tests. Conclusions : The rates of correct antigen identification and of unacceptable crossmatch were similar to those of previous surveys, which were considered satisfactory. The Korean proficiency survey program may have contributed to a high quality of HLA tests today and should be continued for further improvements of the tests tomorrow.

Background : Human leukocyte antigen (HLA) typing based on polymerase chain reaction (PCR) is rapidly replacing the conventional serological method. This study was intended to evaluate BioSewoomTM HLA-A, -B, -C PCR/SSP kit (BioSewoom SSP) which had recently been developed in Korea. Methods : A total of 158 samples from domestic (21) and international (137) HLA proficiency testing (PT) were genotyped with BioSewoom SSP, and its results were compared to consensus results. For comparison with INNO-LiPA HLA-A, -B, -C Typing Kit (INNO-LiPA, Innogenetics, Belgium), 20 samples of Koreans were genotyped with both kits for each HLA-A, -B, -C locus. Results : Among the 21 samples of domestic PT, BioSewoom SSP showed ambiguities as follows: 4 samples (19.0%) in HLA-A, 6 (28.6%) in HLA-B, and 1 (4.8%) in HLA-C. The ambiguities could be resolved by considering the allele distribution of Koreans. Among the 137 samples from international PT, BioSewoom SSP also showed ambiguities as follows: 12 samples (8.8%) in HLA-A, 26 (19.0%) in HLA-B and 6 (4.4%) in HLA-C. Considering the allele distribution of Koreans, the serologic equivalents obtained from BioSewoom SSP showed a full agreement with those of INNO-LiPA in all the loci tested. Twelve (0.007%) among 1,760 PCR reactions from the 21 samples of domestic PT and 20 patient samples produced faint nonspecific bands, but it was negligible. PCR failure of internal control just barely occurred (15 PCR reactions, 0.009%), but it had no bearing on allele assignment. Conclusions : The performance of BioSewoom SSP was comparable to that of INNO-LiPA. All the ambiguities could be resolved by considering the allele distribution of Koreans. It is concluded that BioSewoom SSP has good performance to be used in routine HLA laboratories. (Korean J Lab Med 2007;27:360-8)

Background : In this study, we evaluated the performance of Elite microplate analyzer (Bio-Rad Laboratories, France) and the related assays (ULTRA line) for the detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Methods : Seroconversion panels, HBsAg positive/HBsAg negative (n=99/n=1,422), anti-HCV positive/negative (n=97/n=1,670), and anti-HIV positive/negative (n=112/n=1,704) samples were used to evaluate the performance of Monolisa HBsAg ULTRA, Monolisa HCV Ag-Ab ULTRA, and Genscreen ULTRA HIV Ag-Ab, respectively. The agreement of Elite microplate analyzer with CODA analyzer (Bio-Rad Laboratories, CA, USA) was also evaluated. Results : The detection limit of Monolisa HBsAg ULTRA was 0.034 IU/mL. For Monolisa HBsAg ULTRA, Monolisa HCV Ag-Ab ULTRA, and Genscreen ULTRA HIV Ag-Ab, diagnostic sensitivities were all 100%, diagnostic specificities were 100%, 99.8% and 99.9%, and total CVs (coefficients of variation) were 13.8-17.5%, 3.4-5.2%, and 7.5-9.5%, respectively. The agreement of Elite microplate analyzer with CODA analyzer was 99.5%. Conclusions : The performance of Elite microplate analyzer and the related assays on analytical sensitivity, precision, early detection, diagnostic sensitivity and specificity was all adequate for a mass screening. However, further large multi-center studies should be performed to validate our results. (Korean J Lab Med 2006;26:436-41)

Background : Hepatitis B virus (HBV) DNA quantification is important for the management of HBV infection and identification of the development of resistance. The susceptibility to contamination and more variable reproducibility of results with the conventional HBV DNA quantification method have raised the need of a more simple and accurate method for HBV DNA quantification. Real-time quantitative PCR assays recently introduced in the laboratory can meet these needs. In this study, we evaluated the performance of the Real-Q HBV Quantification kit developed in Korea. Methods : We evaluated the recovery of DNA extraction, the interference of internal control, an analytical sensitivity, specificity, and reproducibility, a clinical specificity, and a reportable range of the Real-Q HBV Quantification kit. The quantification result was also compared to that obtained by the Digene Hybrid-Capture II. Results : The mean percent recovery was 108.6% and there was no interference with the internal control on DNA extraction. None of HIV, hepatitis C virus, or cytomegalovirus showed a cross-reactivity with HBV. This assay detected HBV DNA in a linear range from 10 2 to 10 10 copies/mL, with the detection limit of 56 copies/mL. The assay exhibited a low within-run CV (coefficient of variation) (8.711.9%), between-run CV (10.5-14.7%), and between-day CV (13.2-21.4%). No HBV DNA was detected in any of 100 samples without HBV, resulting in a clinical specificity of 100%. The levels of HBV DNA showed a good correlation with those determined with Digene Hybrid-Capture II (R2 =0.9827). Conclusions : The Real-Q HBV Quantification kit showed a good analytical sensitivity, specificity, and high reliability with a broad reportable range. This assay should be clinically useful in managing patients with HBV infection. (Korean J Lab Med 2006;26:442-8)

Background : The third generation anti-hepatitis C virus (HCV) enzyme immunoassay (EIA) is now in use for screening HCV infection. The aim of this study was to pool the data on the sensitivity and specificity of third generation anti-HCV EIA tests after evaluating the quality of the studies using Standards for Reporting of Diagnostic Accuracy studies (STARD) checklist. Methods : We searched MEDLINE and PubMed databases using keywords about the accuracy of diagnostic tests for HCV infections. Methodological quality was assessed by two persons with a modified STARD checklist. A heterogeneity test was performed, and in case heterogeneity was present, a sub-group analysis was done. Fixed-effects model was used to obtain pool sensitivity and specificity with 95% confidence intervals (CI). Results : A total of 41 studies from 16 papers were selected. The quality score ranged from 6 to 13 (median 10.5); Inter-observer agreement was 93.62% (k=0.69); and 41 studies revealed heterogeneity. We performed a sub-group analysis with only 28 studies from 13 papers that were evaluated to be of high quality. A subgroup using polymerase chain reaction as the reference test revealed homogeneity and was calculated the pooled sensitivity and specificity of 99.92% (CI 99.77-100.07%) and 99.66% (CI 99.45-99.86%) respectively. Studies on test kits with an increased reactivity to the core region also showed homogeneity in sensitivity and the pooled sensitivity was 99.78% (CI 99.53-100.03%). Conclusions : For the first time in Korea, the diagnostic accuracy of test kits was evaluated by meta-analysis using STARD checklist. The methodology shown in this study should help extending laboratory medicine to an evidence-based medicine.

Background : Many studies evaluating the performance of in vitro diagnostic kits have been criti-cized for the lack of reliability. To attain reliability those evaluation studies should be preceded bysample size calculation ensuring statistical power. This study was intended to develop a web-basedsystem to estimate the sample size, which was often neglected because it would require expert knowl-edge in statistics.Methods :For sample size calculation, we extracted essential parameters from the performancestudies on the 3rd generation anti-hepatitis C virus (HCV) kits reported in the literature. We developeda system with PHP web-script language and MySQL. The statistical models used in this system wereas follows; one sample without power consideration (model 1), one sample with power consideration(model 2), and two samples with power consideration (model 3).Results :Among the articles published between 1989 and 2005, 13 articles that evaluated theperformance of anti-HCV kits were identified by searching with Medical Subject Headings (MeSH).The diagnostic sensitivity was 83-100% with a median of 145 samples (range; 12-1,091) and thespecificity was 97-100% with a median of 1,025 samples (range; 33-4,381). The estimated samplesize would be 280 in the model 1, 817 in the model 2, and 1,510 in the model 3, when we set 2%prevalence of HCV infection, 95% sensitivity of a conventional kit, 97% sensitivity of a new kit , 95%significance level (two-sided test), 2% allowable error, and 80% power.Conclusions :Our study indicates that an insufficient sample size is still a problem in performanceevaluation. Our system should be helpful in increasing the reliability of performance evaluation byproviding an appropriate sample size. (Korean J Lab Med 2006;26:299-306)Key Words