Genotyping for RhD and RhCEce Antigens Using Free Circulating Nucleic Acids in Plasma and Serum / 대한수혈학회지

BACKGROUND: The Rh blood group includes several antigens, of which D, C, E, c, and e are clinically important. Although nucleic acids from whole blood can be used for Rh blood group genotyping, it is also possible to genotype free circulating fetal nucleic acids from plasma and serum. We performed Rh blood group phenotyping and genotyping using nucleic acids from whole blood and free circulating nucleic acids from plasma and serum, respectively. The results were compared. METHODS: Forty-four blood samples were phenotyped and genotyped for RhD and RhCE blood groups. Phenotyping was performed by hemagglutination assay. Further tests were performed on RhD-negative samples. Nucleic acids were extracted from whole blood, plasma, and serum. Plasma and serum were prepared after filtration and genotyped by real-time polymerase chain reaction. RESULTS: RhD blood group results showed one (2.3%) discrepant case in which the DEL phenotype appeared wild RHD genotype. Among nucleic acids, there were seven discrepant results: two from plasma and five from serum based on whole blood nucleic acids. RhCE blood group results showed three (6.8%) phenotype-genotype discordances. Among nucleic acids, seven (15.9%mpared to phenotypes. Kappa coefficients of serum were lower than those of plasma. CONCLUSION: RHD and RHCE genotype could be identified by assaying free circulating nucleic acids in plasma or serum. This study suggests that plasma is more reliable than serum as a specimen for RHD and RHCE genotyping of free circulating nucleic acids.

Performance Evaluation of the CAPILLARYS 2 FLEX Piercing Analyzer for HbA1c Determination

BACKGROUND: The hemoglobin A1c (HbA1c) level is widely used to monitor glycemic control in diabetes mellitus patients, and various methods are used for its determination. The CAPILLARYS 2 FLEX Piercing (Sebia) is a fully automated, high-throughput glycohemoglobin (HbA1c) analyzer based on capillary electrophoresis. METHODS: The analytical performance of the CAPILLARYS 2 FLEX Piercing analyzer was evaluated for its precision, linearity, correlation with the Variant II Turbo (Bio-Rad Laboratories, Inc.) analyzer, and its vulnerability to interference by carbamylated hemoglobin. We also investigated its agreement with National Glycohemoglobin Standardization Program (NGSP) targets. All evaluations were performed according to CLSI guidelines EP05, EP06, and EP09. RESULTS: The coefficients of variation (CVs) for within-run and total imprecision were 1.7% and 1.8% at low concentrations and 1.2% and 1.3% at high concentrations, respectively. Linearity was excellent, with R2=0.9882 in the range of 5.13-13.83%; these results highly correlated with those produced by Variant II Turbo (R2=0.9978). The 95% confidence interval (for differences from the NGSP target) was -0.3618-0.3343%. No significant interference of carbamylated hemoglobin was noted. CONCLUSIONS: The CAPILLARYS 2 FLEX Piercing analyzer showed excellent precision and linearity. Its results correlated with those obtained by the Variant II Turbo analyzer, and were agreement with the NGSP target. Therefore, its analytical performance is satisfactory for diabetes diagnosis and treatment monitoring.

Human Platelet Antigen Genotyping Using a Multiplex Single-Base Primer Extension Reaction in Koreans / 대한수혈학회지

BACKGROUND: Alloimmunization of human platelet antigens (HPA) is associated with clinically significant disease, such as platelet refractoriness, neonatal alloimmune thrombocytopenia, or posttransfusion purpura. It is determined by single nucleotide polymorphism of genes for platelet membrane glycoprotein. To date, approximately 27 HPAs have been discovered, and their frequencies differ depending on ethnicity and country. METHODS: We conducted an investigation of prevalence of HPA in the Korean population using a multiplex single-base primer extension reaction (SNaPshot). With 84 specimens from healthy donors, HPA genotyping was performed on 11 different HPAs, including HPA-1, -2, -3, -4, -5, -6, -7, -8, -9, -13, and -15. RESULTS: A total of 90 blood samples were genotyped. The genotype frequencies of HPA were as follows: HPA-1a/1a: 100.0%, -2a/2a: 83.3%, -2a/2b: 14.3%, -2b/2b: 2.4%, -3a/3a: 39.3%, -3a/3b: 52.4%, -3b/3b: 8.3%, -4a/4a: 100.0%, -5a/5a: 95.2%, -5a/5b: 4.8%, -6a/6a: 94.0%, -6a/6b: 6.0%, -7a/7a: 100.0%, -8a/8a: 100.0%, -9a/9a: 97.6%, -9a/9b: 2.4%, -13a/13a: 100.0%, -15a/15a: 23.8%, -15a/15b: 51.2%, and -15b/15b: 25.0%. CONCLUSION: The SNaPshot assay was employed for detection of SNPs in various clinically significant HPA genes. In addition to well-known frequencies of previously reported HPA-1 to -8, this study showed frequencies of HPA-9, -13, and -15 in Koreans for the first time. The SNaPshot technique might be suitable for use in actual clinical testing in patients with platelet alloimmunization.

Analysis of Xenotropic Murine Leukemia Virus-Related Virus (XMRV) in Korean Blood Donors in a Medical Center / 대한수혈학회지

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) has been detected in peripheral blood mononuclear cells (PBMNs), therefore, it has been regarded as being infectious and transmittable by transfusion. Thus, we attempted to detect XMRV in blood samples in order to confirm the absence of XMRV from blood donors. METHODS: We achieved 165 blood donors and four chronic fatigue syndrome (CFS) patients. We performed real-time polymerase chain reaction using the LightCycler 480 (Roche, Penzberg, Germany) for the gag and env genes of the XMRV genome. DNA was extracted from peripheral blood samples. We used Uracil-N-Glycosylase in order to prevent contamination and DNA extracted from mouse embryonic fibroblasts (MEF) for amplification control. RESULTS: No XMRV was detected in any of the blood donors in both the gag and env genes. In four CFS patients, amplification was not detected in the gag gene. In two of four CFS patients, amplifications were detected and the melting temperature was in agreement with that of MEF control in the env gene. CONCLUSION: Although XMRV was not present in blood samples from blood donors, this is the first report on XMRV in Korean blood donors. We confirmed the absence of XMRV in Korean blood donors, the same as studies reported in other countries.

Performance Evaluation of the ADAMS A1c HA-8180 Analyzer for HbA1c / 임상검사와정도관리

BACKGROUND: Hemoglobin A1c (HbA1c) levels are widely used to monitor glycemic control in diabetes mellitus patients, and various methods are used for determining HbA1c levels. The ADAMS A1c HA-8180 (Arkray, Inc., Japan) is a fully automated HbA1c analyzer based on high-performance liquid chromatography (HPLC). METHODS: The analytical performance of the ADAMS A1c HA-8180 analyzer was evaluated on the basis of its precision, linearity, correlation with the Variant II Turbo (Bio-Rad Laboratories, USA), and agreement with the National Glycohemoglobin Standardization Program (NGSP) targets. All evaluations were performed according to Clinical and Laboratory Standard Institute (CLSI) guidelines EP05, EP06, and EP09. RESULTS: Coefficients of variation (CVs) for total precision at low and high levels were 0.99% and 1.16%, respectively. The linearity was excellent with R2 = 0.99 in the range of 4.98-15.10%. Its analytical performance was well correlated with that of Variant II Turbo (r = 0.9987). The 95% confidence interval of bias between the NGSP target and the levels measured using the ADAMS A1c HA-8180 was -0.402-0.225. CONCLUSIONS: The ADAMS A1c HA-8180 showed excellent precision, linearity, correlation with Variant II Turbo, and agreement with the NGSP target. Therefore, its analytical performance is satisfactory for diagnosis and treatment monitoring of diabetes.

Three Cases of Anti-K and the KEL Gene Frequency in the Korean Population / 대한수혈학회지

BACKGROUND: Anti-K is one of the most significant unexpected antibodies that cause hemolytic transfusion reactions. Individuals with anti-K have to be transfused with K antigen-negative red cells. Although Koreans rarely have the K antigen, we have detected three cases of anti-K and we analyzed the Kell blood group genotypes for the KEL*1/KEL*2 alleles at the same time. METHODS: We analyzed the KEL*1/KEL*2 allele genotypes from 261 blood donors at Seoul National University Bundang Hospital. Kell genotyping were carried out using polymerase chain reaction (PCR) and restriction enzyme length polymorphism (RFLP). Identification of anti-K was performed using three kinds of methods; 37degrees C albumin, an anti-human globulin phase tube, a bead cassette and a gel card. Three cases of anti-K also underwent PCR with a sequence specific primer (SSP) for Kell genotyping. For comparison, the KEL*1 allele (698C>T) was synthesized by site-directed-mutagenesis. RESULTS: All 261 donors were KEL*2/KEL*2 homozygotes and a digested KEL*1 allele was not found. The three patients with anti-K were also KEL*2/KEL*2 homozygotes and the reactivities of the anti-K identification test were the same. CONCLUSION: The KEL gene frequency for the KEL*1/KEL*2 allele corresponded with that of the Kell phenotype, as was previously reported. We experienced three cases of anti-K and two out of the three were assumed that they had been transfused with the K antigen-positive blood of foreigners. This study revealed that the possibility of anti-K alloimmunization and hemolytic transfusion reactions cannot be excluded in Koreans.

A Case of Good's Syndrome with Weak ABO Reverse Type / 대한수혈학회지

Good's syndrome (thymoma with immunodeficiency) is a rare cause of combined B-cell and T-cell immunodeficiency in adults. We present here a case of Good's syndrome involving a 52 year-old man with an ABO blood group abnormality. He had undergone surgery for thymoma with myasthenia gravis 27 years ago. He also had a history of pulmonary tuberculosis, herpes zoster and pure red cell aplasia. On admission, he was suspected of having pneumonia, and S. pneumoniae was isolated from blood culture. The immunoglobulin levels were markedly decreased. Lymphocyte subset analysis revealed the absence of CD19+ B cells. The result of ABO typing showed a normal strong reaction on the cell typing, but a relatively weak reaction on the serum typing. Therefore, we performed ABO genotyping to confirm his ABO type, which was revealed to be B/O1 . This case suggests that ABO typing should be performed when the diagnosis of Good's syndrome is made. Moreover, Good's syndrome (thymoma with hypogammaglobulinemia) should be considered and evaluated for in patients with a weak ABO reverse type.

Identification of Compound Heterozygous Mutation in a Korean Patient with Alpha 1-antitrypsin Deficiency / 대한진단검사의학회지

Alpha 1-antitrypsin (AAT) deficiency is a genetic disorder that primarily affects the lungs and liver. While AAT deficiency is one of the most common genetic disorders in the Caucasian population, it is extremely rare in Asians. Here, we report the case of a 36-year-old Korean woman with AAT deficiency who visited the emergency department of our hospital for the treatment of progressive dyspnea that had begun 10 years ago. She had never smoked. Chest computed tomography revealed panlobular emphysema in both lungs, which suggested AAT deficiency. The serum AAT level was 33 mg/dL (reference interval: 90-200 mg/dL). Four exons of the SERPINA1 gene, which is responsible for AAT deficiency, and their flanking regions were analyzed by PCR-direct sequencing. The patient was found to have 1 missense mutation (c.230C>T, p.Ser77Phe; Siiyama) and 1 frameshift mutation (c.1158dupC, p.Glu387ArgfsX14; QOclayton). This is the first Korean case of AAT deficiency confirmed by genetic analysis and the second case of a compound heterozygote of Siiyama and QOclayton, the first case of which was reported from Japan.

Serum Ferritin Levels for Normal Hemoglobin Blood Donors in a Korean Population / 대한수혈학회지

BACKGROUND: Serum ferritin is well known as a marker that is reflective of the iron storage status in blood. But only the hemoglobin level is generally included in the current selection criteria for blood donors. Recent studies have continuously shown that the serum ferritin levels are lower for the blood donors, and especially for women and repeat donors with a normal hemoglobin level. It has been suggested that serum ferritin should be included in the blood donor selection criteria. In this study, we examined the serum ferritin status in a Korean population, and we checked the validity of the recent related studies. METHODS: The hemoglobin and serum ferritin levels of the blood donors who visited Seoul National University Bundang Hospital were measured (total donors: 353, males: 252, females: 101). The hemoglobin levels were evaluated according to gender and the serum ferritin level, and the changes in the serum ferritin level were also checked for the repeat donors. RESULTS: The median serum ferritin level was 89.7 ng/mL (2.5~97.5%, 13.0~280.7 ng/mL). As classified by the serum ferritin levels, 9.9% of the males and 38.6% of the females showed low serum ferritin (< or =30 ng/mL) with a normal hemoglobin level. Among the 33 cases of repeat donors, 25 cases showed reduced serum ferritin changes (mean change: 17.4%). CONCLUSION: Similar to other recent studies, we found that the donors with normal hemoglobin could show a low serum ferritin level and especially the women and repeat donors in a Korean population. For the blood donors' safety, it is time to consider that the serum ferritin level should be included in the criteria for blood donor selection.

Performance Evaluation of HbA1c Test on the Toshiba 200FR NEO Using AutoLab HbA1c Reagent / 임상검사와정도관리

BACKGROUND: Hemoglobin A1c (HbA1c) is widely used for the monitoring of glycemic control in diabetes mellitus patients. Various methods are applied for the determination of HbA1c levels. Recently, a novel National Glycohemoglobin Standardization Program (NGSP)-certificated reagent (AutoLab HbA1c, IVD-LAB, Korea) was introduced for use in an automated chemistry analyzer. We evaluated the analytical performance of this immunoturbidimetry reagent and compared it with the ion-exchange high performance liquid chromatography (Variant II Turbo, Bio-Rad Laboratories, Inc., USA) and immunoassay (Cobas Integra 800, Roche Diagnostics, Germany) methods. METHODS: Toshiba 200FR NEO (Toshiba Medical Systems Co., Japan) with the AutoLab reagent was evaluated for precision, linearity, carryover and compared with Cobas Integra and Variant II Turbo. RESULTS: Coefficients of variation (CVs) of within-run imprecision for low and high level were 1.8% and 0.7%, respectively. CVs of within-laboratory imprecision for low and high level were 2.4% and 1.0%, respectively. The linearity was excellent with R2 = 0.99 in the range of 3.05-15.50%. It was well correlated with Variant II Turbo (R=0.9904) and Cobas Integra 800 (R=0.9992). The carryover rate was 0.4%. CONCLUSIONS: The Toshiba 200FR NEO with the AutoLab reagent showed excellent precision and linearity and minimal carryover rate. It was well correlated with the other widely used methodological instruments. It may be used for the diagnosis and the treatment monitoring of diabetes.

Detection of Bacterial Contamination of Platelets Using the Real-time Polymerase Chain Reaction / 대한수혈학회지

BACKGROUND: The risk of transfusion-transmitted bacterial infection has been reported in addition to the risk of transmission of viral disease. Especially for platelets that are stored at 20degrees C~24degrees C with agitation to sustain platelet function. This storage method facilitates bacterial proliferation. Therefore, sensitive and rapid detection of bacteria must be considered for stored platelets. METHODS: Six concentrations of platelets were spiked (1.5 mL) with five different bacteria and were analyzed by the 16S rDNA real-time polymerase chain reaction (PCR) using the ABI PRISM 7500 (Applied Biosystems, Foster, CA, USA) and the LightCycler 2.0 (Roche, Penzberg, Germany). The 16S rDNA gene was analyzed in three lots by real-time PCR reagents with sterile water. Three concentrations of spiked platelets (0.5 mL) with three different bacteria were preincubated in thioglycollate medium at 37degrees C for 8, 12, 16, 20, and 24 hours and then were analyzed by the 16S rDNA real-time PCR. The spiked platelets (0.5 mL) with fast-growing (8 hours of preincubation) and slow-growing (24 hours preincubation) bacteria were analyzed for the minimum incubation time. RESULTS: The average crossing points (Cps) of the five bacteria were 21.2 with the ABI PRISM 7500 and 22.2 in the LightCycler 2.0. All five bacteria (10(1) bacteria/mL) were detected by both instruments. The average Cps of the three lots by real-time PCR was 29.3, 31.5 and 35.6. The contamination levels of the 16S rDNA were different. Fast-growing and slow-growing bacteria, preincubated at 8 and 24 hours, respectively, were detected at levels of 10(1) bacteria/mL. CONCLUSION: The results of this study suggest that slow-growing bacteria can be detected at concentrations of 10(1) bacteria/mL in platelets preincubated with thioglycollate medium, at 37degrees C for 24 hours using platelets segment.

Evaluation of the Performance of the DG Gel Test for Unexpected Antibody Screening and Identification / 대한수혈학회지

BACKGROUND: There is a recent trend of increased use of the gel test for detecting unexpected antibodies because of its simplicity and the ease with which a definitive interpretation can be made. The aim of the present study was to evaluate the diagnostic performance of newly developed DG Gel microtube column agglutination system as compared with the tube method and other two microtube column agglutination systems (DiaMed-ID, Ortho BioVue). METHODS: We collected 305 patients who were screened for unexpected antibody from February to July 2007. These samples were screened and we identified the unexpected antibody with the tube method and three microtube column agglutination systems. RESULTS: The highest estimated sensitivity of the screening test was 97.4% for DiaMed-ID. The highest estimated specificity of the screening test was 100% for DG Gel. The least number of discordant identification results was seven for the DG Gel. CONCLUSION: DG Gel has good diagnostic efficacy and accuracy for identifying unexpected antibody. DG Gel might be used as a replacement or supplement for the previous tests.

Detection of Mycobacterium tuberculosis complex Using Real-time Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: For the detection of Mycobacterium tuberculosis complex (MTB), PCR is known to be sensitive, specific, and rapid compared to the conventional methods of acid-fast-bacilli (AFB) smear and culture. We evaluated a new approach for MTB detection using real-time PCR. METHODS: The specificity of real-time PCR was evaluated using 20 MTB isolates and 37 nontuberculous mycobacteria (NTM) isolates identified by AccuProbe Mycobacterium tuberculosis complex colony identification test (Gen-Probe Inc., USA) and Myco-ID (M&D, Korea). One hundred sputum specimens (50 AFB smear-positive and 50 negative specimens) were analyzed using real-time PCR and Amplicor Mycobacterium tuberculosis test (Roche, Germany). The results of real-time PCR positives (55 samples) and negatives (598 samples) were analyzed by AFB smear and culture. RESULTS: The real-time PCR assay accurately discriminated between MTB and NTM species. Realtime PCR and Amplicor test yielded the same results in 96.0% (96/100) of the sputum specimens tested. The sensitivity and specificity of real-time PCR based on AFB culture were 97.4% and 88.5%, respectively. Of the 55 real-time PCR positive specimens, 83.6% (46/55) were culture-positive, 30.9% (17/55) were smear-positive, 52.7% (29/55) were smear-negative and culture-positive, and 14.5% (8/55) were both smear and culture-negative. Among the 598 real-time PCR negative specimens, 60 were not tested for AFB smear or culture and 10 were contaminated. Of the remaining 528 specimens, 478 (90.5%) were both smear and culture-negative and 39 (7.4%) were culture-positive. CONCLUSIONS: For the detection of MTB, real-time PCR was sensitive and specific and comparable to conventional methods. It can be used for rapid identification of M. tuberculosis in clinical laboratories.

A Case of ABO*Ael02/O04 Genotype with Typical Phenotype O / 대한진단검사의학회지

Ael is a rare blood type which has the least amount of A antigen among A subgroups. It can be detected by special tests performed to resolve the discrepancy between red cell and serum typing in routine serological typing. The presence of A antigen on Ael red cell is demonstrable only by adsorption and elution tests. An Ael individual does not secret A substance in the saliva and may have anti-A antibody in the serum which is usually less reactive with the reagent red cells than anti-B antibody. In Korea, Ael02 has been reported more frequently than other Ael alleles. We report a case of Ael02/O04 who presented as typical phenotype O with strong anti-A and anti-B antibodies and no A antigen detected even by adsorption and elution tests. The case has been proved to be Ael02/O04 by direct sequencing analysis. In individuals with history of discrepancies in the results of ABO phenotyping, ABO genotyping is needed for an accurate evaluation of their blood type.

Determination of the RhC/c Blood Group by Polymerase Chain Reaction with Sequence-specific Primers of the Intron 2 Insert of the RHCE Gene / 대한수혈학회지

BACKGROUND: RhC/c blood group antigens are of clinical importance and molecular genotyping for them can be useful when serological typing is difficult. A method to determine the RhC/c genotype, by targeting exon 1 nt48 and exon 2 nt307, has been used. However, this approach is not accurate for the RHc(cyt48) variant allele. We applied a more accurate genotyping method, using the intron 2 109 bp insert of the RHCE gene, and evaluated its performance in comparison with the standard method. METHODS: RhD and RhC/c serotypes of 236 subjects were determined. We compared two genotype results with the serological phenotype. One method examined the allele-specific exon 1 nt48 and exon 2 nt307 polymorphism area (Method 1), while the other method detected the intron 2 insert instead of the exon 1 nt48 (Method 2) by polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: The predicted phenotypes by Method 1 were not matched with the true phenotypes in 24 cases (24/236, 10.2%). By contrast, the predicted results by Method 2 matched with true phenotypes in all cases except one. The RHc(cyt48) variant was suspected in 22 cases (23.7%) of the 93 Rhc cases. CONCLUSION: For the determination of the RhC/c genotype in Koreans, the method that analyzes exon 1 nt48 is inaccurate. Instead, intron 2 insert analysis with exon 2 nt307 by PCR-SSP appears to be a more accurate alternative.

Analysis of Partial D Subtypes by Various Anti-D Reagents / 대한수혈학회지

BACKGROUND: There are some previous reports about partial D in Korea. However, the frequency of the partial D in Korea is still unknown. In this study, subtypes of partial D were analyzed by the use of various commercially available anti-D reagents. METHODS: We collected 273 cases determined as RhD negative by RhD typing using the tube method with monoclonal IgM/IgG anti-D reagent (Bioscot. Livingston, UK) from 80,062 cases that were screened between January 2004 and August 2007. The cases were divided into three periods (I, II, III), according to the manufacturers and numbers of anti-D reagents used. A weak D test was performed by using the tube method with various anti-D reagents. The cases with different reactivity between anti-D reagents were determined as partial D, and further analyzed the subtypes by reactivity patterns according to the target epitope of anti-D reagents. An additional test using the ID-Partial D Typing Card (DiaMed, Cressier, Switzerland) was conducted during period III. RESULTS: Five cases showed reactivity patterns of weak D and 16 cases showed reactivity patterns of partial D. Ten cases of partial D were typed as DVI and three cases were typed as DFR. During period III, five cases were typed as DVI and one case was typed as DFR. These results were different from the results obtained with the use of the ID-Partial D Typing Card. CONCLUSION: DVI, which is the most common subtype of partial D, is also common in Korea. Therefore, RhD typing and a weak D test should be performed using combined anti-D reagents that enable the differentiation of DVI from other subtypes.

The Effect of Number of Reagent Red Cells on the Antibody Screening and Identification / 대한수혈학회지

BACKGROUND: The number of reagent red cells has an effect on the results of an unexpected antibody screening test. We evaluated the effect of the number of reagent red cells on antibody screening and identification using test panels of a DG Gel (Diagnostic Grifols, Barcelona, Spain). METHODS: A total of 310 samples were tested in parallel using SeraScan Diana 2 and SeraScan Diana 4 (Diagnostic Grifols, Barcelona, Spain) and ID-DiaCell (DiaMed, Cressier, Morat, Switzerland). Positive samples as determined by the use of SeraScan and ID-DiaCell were identified on an ID-Dia panel (DiaMed), Identisera Diana and Identisera Extend (Diagnostic Grifolsn). RESULTS: Among the 310 samples, 54, 59 and 59 samples were determined as antibody positive by the use of SeraScan Diana 2, SeraScan Diana 4 and ID-DiaCell, respectively. Unexpected antibodies were identified in 10/59 samples (17%) by the use of SeraScan Diana 4, and were identified in 34/59 samples (57.6%) by the combined use of SeraScan Diana 2 and SeraScan Diana 4. Identification of unexpected antibodies by the use of Identisera Diana or Identisera Extend was not different. CONCLUSION: When the results of SeraScan Diana 2 and SeraScan Diana 4 are integrated, unexpected antibodies could be identified in 57.6% of the screening-positive samples. Therefore, if the number of reagent red cells is increased, some antibodies can be identified by antibody screening tests, and the results can be used to validate those of antibody identification tests.

ABO Genotyping using a Multiplex Single-base Primer Extension Reaction / 대한수혈학회지

BACKGROUND: ABO genotyping is being widely used in the case of ABO discrepancies and in forensic medicine. We have designed a method using a multiplex single-base primer extension reaction that has allowed us to detect six single nucleotide polymorphism (SNP) sites in the ABO gene and to determine ABO genotypes. METHODS: Genomic DNA was isolated from the peripheral blood of 75 unrelated Korean subjects. Exon 6 containing nucleotides 261 and 297 and exon 7 containing nucleotides 703, 802, 803 and 1059 were amplified using two pairs of primers. Using the products as templates, a multiplex single-base primer extension reaction was performed with six typing primers of different lengths for the six SNP sites. These reactions were performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 3130xl Genetic Analyzer (Applied Biosystems). RESULTS: The ABO genotypes determined by this method (75/75) all matched the genotypes that were determined by the use of the polymerase chain reaction using sequence-specific priming (PCR-SSP). We analyzed the peak pattern detected at each of the six SNP sites for each sample. For the smaller-sized primers, peaks were shifted to the right-side compared with the expected site and for the larger-sized primers peaks was close to the expected site. In addition, the coefficients of variation (CVs) of the smaller-sized primers were higher than the CVs of the larger-sized primers. CONCLUSIONS: We are able to detect six SNP sites in the ABO gene and to determine ABO genotypes using a multiplex single-base primer extension reaction.

Analysis for Eight ABO Alleles in Korean Population / 대한진단검사의학회지

BACKGROUND: ABO genotyping is a useful tool in case of ABO discrepancies and in legal medicine. Recent knowledge of various alleles in the ABO gene has led to the need of a different method that can cover numerous polymorphisms. We performed a polymerase chain reaction using sequencespecific priming (PCR-SSP) with 12 primer sets and evaluated its value in the detection of 8 ABO alleles. METHODS: Genomic DNA was isolated from peripheral blood of 222 unrelated Koreans. Sequencespecific primer sets for the nucleotides 261, 297, 467, 802, 803, and 1059 were selected, and 12 PCR reactions were performed for each sample. Direct sequencing was performed to evaluate the accuracy of discrimination between A1(Pro) and A1(Leu) and between O1 and O1v. RESULTS: All the ABO genotype patterns were in an exact match with the ABO phenotypes. The results from sequencing and PCR-SSP were equivalent. The allele frequencies of A1, B, O1, and O1v were 27.25%, 19.82%, 27.25%, and 25.68% respectively. Out of total 121 A1 alleles, 6 (4.96%) were A1(Pro) alleles and 115 (95.04%) were A1(Leu) alleles. No A2, O2, or CisAB alleles were found in this study. CONCLUSIONS: The proportion of O1v allele was similar to that of O1 allele. This was an unexpected result. We developed a method for detecting 8 ABO alleles by PCR-SSP; the method was accurate and was able to discriminate between A1(Pro) and A1(Leu) and between O1 and O1v.

Detection of Enterovirus in Cerebrospinal Fluid by Real-Time Nested Reverse Transcription Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: Enterovirus is a common cause of aseptic meningitis, respiratory disease and nonspecific febrile illness. The conventional methods for laboratory diagnosis of enterovirus infections have been virus culture and serotyping by an immunofluorecent test. We studied a new and more rapid approach for enterovirus detection in cerebrospinal fluid (CSF) by real-time nested PCR. METHODS: This study was performed on 50 CSF specimens from patients suspected of aseptic meningitis. Enterovirus was detected in CSF by PCRs for 3 different targets and real-time nested PCR. Enterovirus culture was also performed in 44 CSF specimens. RESULTS: The positive rate of PCRs for each of the 3 different targets was 26.0%, 40.0%, or 46.0%, and that of real-time nested PCR was 86.0%. Only 6.8% were positive in culture. Thus, the positive rate of real-time nested PCR was much higher than other methods. CONCLUSIONS: Our study revealed that the real-time nested PCR should be useful for diagnosis of enterovirus infections because of a high sensitivity and rapid detection.