A Dispermic Chimera with Mixed Field Blood Group B and Mosaic 46,XY/47,XYY Karyotype

Chimerism in humans is a rare phenomenon often initially identified in the resolution of an ABO blood type discrepancy. We report a dispermic chimera who presented with mixed field in his B antigen typing that might have been mistaken for the B3 subtype. The propositus is a healthy Korean male blood donor. Neither his clinical history nor initial molecular investigation of his ABO gene explained his mixed field agglutination with murine anti-B. Chimerism was suspected, and 9 short tandem repeat (STR) loci were analyzed on DNA extracted from blood, buccal swabs, and hair from this donor and on DNA isolated from peripheral blood lymphocytes from his parents. The propositus' red blood cells demonstrated mixed field agglutination with anti-B. Exon 6 and 7 and flanking intronic regions of his ABO gene were sequenced and revealed an O01/O02 genotype. B allele haplotype-specific PCR, along with exon 6 and 7 cloning and sequencing demonstrated a third ABO allele, B101. Four STR loci demonstrated a pattern consistent with a double paternal chromosome contribution in the propositus, thus confirming chimerism. His karyotype revealed a mosaic pattern: 32/50 metaphases were 46,XY and 18/50 metaphases demonstrated 47,XYY.

Serologic Variability of the A(var) (784G>A) and Its Property of Different Expression Depending on Co-inherited ABO Allele / 대한수혈학회지

BACKGROUND: A allele, A(var), characterized by a 784G>A polymorphism (Asp262Asn) has been identified only in Korean A(weak)B donors. This study evaluated the serological and genetic characteristics of thirteen samples with newly identified A(var) allele. METHODS: This study examined 10 samples with the A(var) allele including 4 members from a family, who were randomly obtained from blood donors recruited at Gwangju-Chonnam Red Cross Blood Center, and patients at the Chonnam National University Hospital. Routine ABO serologic tests, ABO genotyping using an allele specific polymerase chain reaction (AS-PCR), and the sequencing of exon 6 and 7 of ABO gene were performed on all samples. In addition, sequencing of exon 1~5 of the ABO gene was carried out on two randomly selected samples. RESULTS: The A(var) allele was identified in nine A(weak)B and one O (II-1 of the family study) sample. Eight of these nine individuals showed 1+ agglutination with the monoclonal anti-A reagents on forward typing but one sample showed no agglutination. Weak anti-A was detected in all sera. From the family study, the A(var) allele, which was transmitted from the propositus through her descendant (II-1, II-3 and III-1), produced either the weak A phenotype when inherited with a B allele or the O phenotype when inherited with an O allele. CONCLUSION: A(var) erythrocytes showed different agglutination patterns to anti-A. Different expressions (possible allelic enhancement) were observed depending on the co-inherited ABO alleles from samples with the A(var) allele.

A Family Study of Ael02 Allele Expressing Different Phenotypes Depending on Co-inherited ABO Alleles / 대한수혈학회지

Compared with A101, Ael02 is characterized by 467C>T, 646T>A and 681G>A polymorphisms, resulting in two amino acid substitutions (Pro156Leu and Phe216Ile). The first study in Korea was reported at 2003. However, only unrelated donors were characterized. This study carried out molecular genetic analysis of a 26 year-old male propositus diagnosed with the Ael subgroup by serological tests along with his family. The propositus had the genotype Ael02/B101 expressing the AelB phenotype, and his father the genotype Ael02/O01 expressing the O phenotype. These findings suggest that the AelO2 allele is expressed as different phenotypes depending on the co-inherited ABO alleles.

A Patient with RhD(el) (1227G>A) Failed to Produce Detectable Anti-D after Transfusion of RhD Positive Red Blood Cells / 대한수혈학회지

Only 0.15% of all donors in Korea are RhD negative, which has led to a chronic shortage of RhD negative blood. Most physicians are aware of the potential for RhD alloimmunization after transfusing RhD+ red blood cells into RhD- patients. Hence, the undertransfusion of RhD- patients might be occurring in Korea. A 66-year-old man without a history of transfusion tested negative for anti-D in his serum. In an emergency situation where RhD- blood was unavailable, the patient received two units of RhD+ RBCs. Anti-D was not detected over three months after the transfusion. The red cells of the patient showed no agglutination with the anti-D reagent and a negative result by the standard weak D test. The polymerase chain reaction-sequence specific primers (PCR-SSP) and sequencing revealed D(el) (1227G>A) in the patient.

Resolution of ABO Discrepancies by ABO Genotyping / 대한진단검사의학회지

BACKGROUND: Before a blood transfusion, both red cell and serum typing need to be matched for ABO tests on the donor and patient (recipient). When a mismatch exists in the tests, additional ABO genotyping and serological tests are required for the resolution of the discrepancy. We performed ABO genotyping on a series of blood donors and patients with ABO discrepancies to assist in resolving their blood groups. METHODS: We examined 46 samples with ABO discrepancies from a random pool of donors recruited at Gwangju-Chonnam Red Cross Blood Center and from patients at Chonnam National University Hospital between May 2004 and July 2005. ABO genotyping was performed on all samples with an allele specific polymerase chain reaction for differentiation of A, B,O, cis-AB, A(var) (784 G>A), and B(var) (547 G>A) alleles; routine serologic tests were also performed. Exon 6 and 7 of ABO gene from five samples were sequenced. RESULTS: The genotypes of 18 donors/patients with weakened A or B antigen expressions consisted of 4 cases of cis-AB/O (3 A(2)B(3), 1 A(2)B); 5 cases of cis-AB/A (5 A(1)B(x or el)); 2 cases of A/O (1 O, 1 A(m or x)); 1 case of B/O (1 B(m or x)); 4 cases of A/B (1 A(2)B , 1 A(1)B(x or el), 2 A(1)B(3)); and 2 cases of A(var)/B (2 A(w)B). On the other hand, the genotypes of 28 samples with unexpected serum reactions included 18 cases of A/O (16 A(1), 2 A(int)); 7 cases of A/A (5 A(1), 1 A(1)B(x or el), 1 A(1)B(w)); and 3 cases of O/O (1 O, 2 B(w)). CONCLUSIONS: ABO genotyping is useful for differentiating the ABO discrepancies that were difficult to resolve by serological tests. The most frequent unusual red cell reactions were weak A and B antigen expressions, which were resulted from the ABO subgroup alleles including cis-AB allele, whereas the most frequent unusual serum reactions were caused by decreased anti-B titers.

Analysis of Complete Exons and Flanking Introns of ABO Gene in Korean B(3) Blood Donors / 대한수혈학회지

BACKGROUND: There have been several studies aimed at determining the presence of the B(3) specific alleles in Korean B(3) blood donors. However, in these samples, only consensus exons 6 and 7 have been detected. Therefore, this study analyzed the complete exons (1~7) and flanking intronic region of the ABO gene sequence in B(3) donors. METHODS: A total of 12 B(3) blood donors collected at the Gwangju-Chonnam Red Cross Blood Center were identified using standard tube techniques. The genomic DNA was isolated from the peripheral blood and of exons 1~7 including flanking intronic regions were sequenced and an allele specific polymerase chain reaction (AS-PCR) was performed. RESULTS: Complete exon and flanking intronic analysis of the ABO alleles revealed the consensus B101 allele along with either the O01 or O02 allele in 11 out of the 12 donors. The remaining 1 donor had the Bw03/O01 genotype. CONCLUSION: No B(3) specific novel alleles were found in most Korean B(3) donors, and the genetic basis of B(3) blood group could not be explained.

A Simplified ABO Genotyping by Allele-Specific Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: Genotyping of ABO gene could be more informative and valuable than serological typing in some situations such as the resolution for ABO discrepancy between the cell typing and serum typing and determination of A and B subgroups. We developed a simple allele-specific polymerase chain reaction (AS-PCR) method without the use of any restriction enzymes to detect the A, B, O, and cis-AB alleles for Koreans. METHODS: An AS-PCR was designed with amplification refractory mutation system (ARMS) at nt (nucleotide) 261 (exon 6) and at nt 526, 803 (exon 7) of ABO gene to detect specific nucleotide sequence differences between the ABO alleles. We tested for ABO genotyping 60 DNA samples previously tested by PCR-RFLP and stored at -70degreeC. These samples had been obtained from blood donors recruited at the Gwangju-Chonnam Red Cross Blood Center between July 2002 and February 2003. RESULTS: With our new PCR method, the genotypes of the 60 samples were found to be A/O (n=10), A/A (n=5), B/O (n=10), B/B (n=5), O/O (n=10), cis-AB/A (n=5), cis-AB/B (n=5), and cis-AB/ O (n=10), which were the s ame results obtained previously with PCR-RFLP. CONCLUSIONS: Our AS-PCR is a simple and accurate method for the detection of A, B, O, and cis-AB alleles for Koreans.

A Case of A1B3 Child from a Group A Mother and a Group B Father: New Group B Allele Arising from 547G>A / 대한수혈학회지

Group B subtype, A1B3, was observed in a 22-year-old blood donors by conventional serologic test. In our family study, his father demonstrated uncomplicated B phenotype and his mother typed as group A. We sequenced exon 6 and 7 of phenotypically A1B3 propositus and his family members by direct sequencing and PCR-based cloning. And we have identified a novel Bvar allele characterized by a 547G>A polymorphism present in propositus and his father. This suggests that the Bvar allele is expressed differently depending on the co-inherited ABO allele.

Evaluation of Spectrophotometric Broth Microdilution Methods for Determining Antifungal Susceptibilities of Aspergillus Species / 대한진단검사의학회지

BACKGROUND: A spectrophotometric approach to minimum inhibitory concentration (MIC) determination for filamentous fungi may provide an objective and rapid MIC reading, and quantify the hyphal growth of molds. In this study, we evaluated two spectrophotometric broth microdilution methods (SBM) to determine amphotericin B and itraconazole MICs for Aspergillus species isolated from clinical specimens. METHODS: A total of 80 clinical isolates (20 A. fumigatus, 20 A. flavus, 18 A. niger, 20 A. terreus, and 2 A. nidulans) were tested for amphotericin B and itraconazole susceptibility by the broth microdilution method. The MIC endpoint was calculated by the spectrophotometer with microplate reader (SBM-Spec method) or colorimetric XTT (tetrazolium dye) method (SBM-XTT method). The results of the SBM method were compared with those of NCCLS M38-A broth microdilution method. RESULTS: The MICs of amphotericin B by the NCCLS M38-A method ranged from 0.125 to 8 g/mL, and those of itraconazole ranged from 0.25 to 2micrograms/mL. The agreement of SBM-Spec and SBM-XTT methods within one dilution of the NCCLS M38 reference were 98.8% and 96.3% for the ampho-tericin B, and 98.8% and 100% for itraconazole, respectively. The agreements between SBM-Spec and SBM-XTT methods were 97.5% for amphotericin B and 98.8% for itraconazole. CONCLUSIONS: In antifungal susceptibility testing of Aspergillus species, the SBM method includ-ing SBM-Spec and SBM-XTT methods showed high levels of agreements with the NCCLS M38-A method. The SBM methods can be useful in the clinical laboratory.

Molecular Typing of Candida parapsilosis Isolates from Patients and Healthcare Workers by Pulsed-Field Gel Electrophoresis / 감염

BACKGROUND: The epidemiology of Candida parapsilosis is still undefined and may involve sources such as hospital environment and hands of healthcare workers (HCWs). We performed molecular typing of C. parapsilosis isolates from intensive care unit (ICU) patients and to compare these with isolates from ICU HCWs. METHODS: A total of 57 C. parapsilosis strains including isolates from blood (n=20) and central venous catheter (n=14) of patients and isolates from hands of HCWs (n=23) were analyzed. All the isolates were collected from candidemic patients (n=20) and HCWs (n= 18) of two ICUs during January 1999 to December 2000. Pulsed-field gel electrophoresis (PFGE) analysis were performed by electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA using SfiI. RESULTS: PFGE separated 57 isolates into 37 distinct types. For bloodstream isolates, a total of 18 different DNA types were identified among 20 isolates from 20 .patients: two strain types (K1 and K13) were shared by four isolates from four patients. The catheter strains from each patient exhibited the same PFGE pattern with bloodstream isolates. Of 23 strains from 18 HCWs, a total of 20 different DNA types were identified: 3 strain types shared by 6 isolates from 6 HCWs. Only one of the PFGE types of the HCWs was shared with patient isolates; an isolate with the same K13 pattern as isolates of two patients was found the hands of HCW. CONCLUSION: This suggest that although C. parapsilosis isolates have a high level of genetic diversity, nosocomial transmission may occur among ICU patients and HCWs via hands.

Random Amplified Polymorphic DNA (RAPD) Analysis for Aspergillus Species Isolated from Clinical Specimens / 대한임상미생물학회지

BACKGROUND: Aspergillus species are second only to Candida species as the most commonly isolated fungi from clinical specimens. As well as the identification of the Aspergillus species, it has been necessary for epidemiological studies to differentiate between strains of the same species. We performed genotypic identification and characterization of species and strains within the genus Aspergillus by using RAPD. METHODS: A total of 63 clinical strains of Aspergillus species (including 21 A. fumigatus, 12 A. flavus, 12 A. niger, 12 A. terreus, 3 A. nidulans, and 3 A. sydowii) from 63 patients was analyzed. For RAPD alanysis, M13 primer (5'GAGGGTGGCGGTTCT3') and five random 10-mer primers (OPC-6, 7, 10, 18 and 20; Operon Technologies, USA) were used. RESULTS: The RAPD patterns by M13 primer appeared to be identical when the isolates of the same Aspergillus species were compared. Distinctive and reproducible sets of amplification products by primer M13 were observed for different Aspergillus species: 60 of 63 (95%) isolates were correctly identified by the RAPD analysis using primer M13. RAPD patterns obtained from different strains of the same Aspergillus species by five OPC primers were far more similar than those derived from different Aspergillus species, but the RAPD profiles with some OPC primers showed polymorphism among isolates of the same Aspergillus species. The application of some OPC primers made it possible to cluster the isolates of the same Aspergillus species into several groups. CONCLUSION: These results indicate that RAPD can be useful for the rapid identification of Aspergillus species and for strain typing in the epidemiological investigations.

Random Amplified Polymorphic DNA (RAPD) Analysis for Candida parapsilosis Isolates from Blood Cultures / 감염

BACKGROUND: Candida parapsilosis is an important nosocomial pathogen that can form biofilms (slime) on prosthetic material and cause catheter- related bloodstream infections. Genetic heterogeneity has been reported within clinical isolates of C. parapsilosis, but clinical significance of these different genotypes is not clear. We investigated random amplified polymorphic DNA (RAPD) genotypes of bloodstream isolates of C. parapsilosis and their relation to slime production. METHODS: Twenty-three bloodstream isolates and 20 strains from other sites were analyzed. For RAPD, five random 10-mer primers were used and the results were analyzed by the numerical taxonomy system and multivariate analysis system (NTSYS-pc). Slime production was evaluated by growing the organism in Sabouraud broth with 8% glucose and examining the walls of the tubes for the presence of an adherent slime layer. RESULTS: RAPD analysis separated 43 isolates of C. parapsilosis into four distinct genotypes. All 23 blood isolates belonged to type I, whereas the isolates from other sites consisted of type I (n=13), II (n=2), III (n=2) and IV (n=3). Eighty-three percent (19/23) of blood isolates were slime positive, whereas 50% (10/20) of isolates from other sites were slime positive. Slime positivity was observed in 81% (29/36) of type I isolates, in contrast to 0% (0/7) in all other types (types II~V). CONCLUSION: We suggest that C. parapsilosis isolates, which produce slime, are possibly of the same or similar RAPD type.

Plasmid DNA and Random Amplified Polymorphic DNA (RAPD) Analyses of Vancomycin-Resistant Enterococcus faecium Isolates / 대한임상병리학회지

BACKGROUND: Vancomycin-resistant enterococci (VRE) have been increasingly reported worldwide. The understanding of VRE dissemination in the hospital requires a molecular typing of the strains. VRE appeared recently in Chonnam University Hospital. The purpose of this study is to analyse the strains for their genetic relatedness. METHODS: Nine vancomycin-resistant E. faecium isolates, collected from six patients during 1995-1996 in Chonnam University Hospital, were typed using plasmid DNA and RAPD analyses. The plasmid DNA of the isolates was obtained by a alkaline lysis method. For RAPD, eight random primers were used. The cluster analysis was performed by NTSYS-pc (numerical taxonomy system and multivariate analysis system, version 1.50, Applied Biostatistics Inc., CA). RESULTS: Nine VRE isolates were separated into two different molecular types (group A and B) by the plasmid DNA patterns, which were agreed with the RAPD results: the isolates of each group showed the same plasmid DNA patterns and high similarity values in the RAPD analysis. Group A was consisted of two strains isolated from two patients who were admitted at the same room in May 1995. Seven strains of group B were isolated from four patients in the different wards during June 1995 to June 1996. CONCLUSIONS: Nine VRE isolates from six patients were typed to two groups by plasmid DNA or RAPD analysis. These results suggested the intrahospital spread of two clonal strains of vancomycin-resistant E. faecium.

Detection of Human Papillomavirus in Uterine Cervical Cancer Tissues by Polymerase Chain Reaction, In Situ Hybridization and Polymerase Chain Reaction In Situ Techniques / 대한임상병리학회지

BACKGROUND: Human papillomavirus (HPV) is a small double-stranded DNA virus. Of HPV, type 16 and 18 are associated with high risk in the development of cervical cancer. In order to evaluate HPV infections, several HPV typing and detection methods have been developed. The aim of this study was to compare the detection rates of HPV 16 and 18 by polymerase chain reaction (PCR), in situ hybridization(ISH), and PCR in situ in uterine cervical cancers. METHODS: PCR, ISH and PCR in situ were performed for the detection of HPV DNA in fifty-one formalin fixed, paraffin embedded blocks of uterine cervical cancer tissues. Twenty uterine cervical specimens from patients with uterine myomas were used as controls. RESULTS: The detection rates of HPV 16 and HPV 18 in cervical cancers were 56.9% (29/51) and 45.1% (23/51) by PCR, 9.8% (5/51) and 5.9% (3/51) by ISH, 17.6% (9/51) and 11.8% (6/51) by PCR in situ, respectively. In control group, the detection rate of HPV 16 and 18 by PCR were 10% (2/20) and 5% (1/20), but HPV was not detected by both ISH and PCR in situ. CONCLUSION: PCR was the most sensitive method for the detection of HPV. However, PCR in situ was more informative fort the specific detection and cell localization of HPV DNA.

Comparison of Third-generation Enzyme-linked Immunosorbent Assays for Detection of Antibody to Hepatits C Virus / 대한임상병리학회지

BACKGROUND: Little Is known about the compared efficiency of different third generation enzyme-linked immunosorbent assays (ELISA) fort the detection of anti-HCV. We examine the relative sensitivity and specificity of three third-generation anti-HCV assays, and results of discrepant samples among the anti-HCV ELISA are compared with data of a third-generation recombinant immunoblot assay and reverse transcription polymerase chain reaction (RT-PCR) . METHODS:A total of 167 samples (61 positive and 106 negative), screened by a second-generation IMx(R) anti-HCV assay (Abbott 2.0; Abbott Laboratories, USA), weve tested with Innotest HCV 3.0(R) (Green Cross, Korea), LG HCD 3.0(R) (LG, Korea) and DONG-A HCV 3.0(R) (Dong-4, Korea). The discrepant specimens among the 4 anti-HCV ELISA were tested by LG HCD Confirm(R) (LG, Korea) and RT-PCR. RESULTS: The concordance rates of all 4 ansi-HCV ELISA were 80.2% (134/167) and 92.2% (154/167), respectively. The 28 and 31 of 33 specimens showing discrepancy among 4 anti-HCV ELISA were tested with LG HCD Confirm and RT-PCR, respectively. Serum HCV RNA was positive in 2 of 2 reactive and in 6 of 26 nonreactive on LG HCD Confirm. The sensitivity, specificity, positive predictive value, negative predictive value and concordance rate of 4 anti-HCV ELISA were 97.7%, 85.2%, 70.0%, 99.0% and 88.5% (Abbott 2.0) ; 81.4%, 96.7%, 89.7%, 93.7% and 92 7% (Innotest 3.0), 81.4%, 98.4%, 94.6%, 93.8% and 93.9% (LG 3.0), 86.0%, 95.7%, 88.1%, 95.1% and 93.3% (DONG-A 3.0), respectively. CONCLUSIONS: These data indicate that the sensitivity and specificity of 3 third-generation anti-HCV ELISA are comparable, and that these reagents demonstrate improved specificity compared to the second-generation ELISA.