Application of bone marrow samples for discrimination of acute promyelocytic leukemia from other types of acute leukemia using the routine automated hematology analyzer.

INTRODUCTION: Unreported parameters produced by automated blood cell counter, particularly large unstained cells (LUC) and delta neutrophil index (DNI), indicated the presence of immature and possibly abnormal cell populations in white blood cell population. The purpose of this study was to investigate the laboratory performance for discrimination of acute promyelocytic leukemia (APL) cells from other types of leukemia cells and clinical value of LUC and DNI parameters in bone marrow (BM) samples of patients with acute leukemia. METHODS: A total of 73 BM samples of patients with various type of acute leukemia were analyzed. LUC and DNI parameters were determined by an automated hematology analyzer (ADVIA 120; Siemens Healthcare Diagnostics, New York, NY, USA). Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney U methods. Receiver operating characteristic curve (ROC) analysis, survival analysis, and Cox proportional hazard model were used to evaluate the clinical implication. RESULTS: There were significant differences (P < 0.05) between APL group and other group in the DNI and LUC values except for DNI between APL group and non-APL myeloid leukemia group. The area under curve of LUC was larger than that of DNI from the ROC analysis for discrimination between APL group and other group. High LUC value was associated with the increased risk of adverse outcomes and the worse overall survival in patients with acute leukemia. CONCLUSION: Delta neutrophil index and LUC in BM showed discriminating power of APL cells from other leukemia cells.

Distinctive hematological abnormalities in East Asian neonates and children with Down syndrome.

INTRODUCTION: Neonates with Down syndrome (DS) are predisposed to developing transient abnormal myelopoiesis (TAM) and acute myeloid leukemia (AML) associated with DS. However, there is a paucity of data on hematological aberrations and GATA1 mutations in neonates with DS in East Asian populations. METHODS: Total 109 patients with DS who had one or more CBCs obtained were enrolled. The molecular analysis of the GATA1 gene performed in 10 patients (three TAM, three AML associated with DS at diagnosis, one remission case of AML associated with DS and three DS without TAM or AML). RESULTS: East Asian DS neonates showed low frequency of thrombocytopenia, uncommon neutrophilia and higher prevalence rate of TAM compared to previous reports from western countries. GATA1 mutations were identified in almost all TAM and AML associated with DS samples, but were not detected in the samples from DS without TAM or AML associated with DS. CONCLUSION: East Asian DS neonates and children showed distinctive spectrum of hematological abnormalities.

Chimerism and mosaicism are important causes of ABO phenotype and genotype discrepancies.

Discrepancies between blood group genotype and RBC phenotype are important to recognize when implementing DNA-based blood grouping techniques. This report describes two such cases involving the ABO blood group in the Korean population. Propositus #1 was a 22-year-old healthy man undergoing pretransfusion testing for minor surgery. Propositus #2 was a 23- year-old male blood donor. RBCs from both propositi were determined to be group AB and demonstrated unusual agglutination patterns on forward typing, which were inconsistent with their ABO genotype determined by allele-specific (AS) PCR. RBCs from propositus #1 demonstrated mixed field agglutination with both anti-A and -B, while RBCs from propositus #2 demonstrated mixed field only with anti-A reagents. Both had B/O genotypes by AS-PCR. Cloning and sequencing of ABO exons 6 and 7 revealed three alleles in both propositi: propositus #1: A102/B101/O04; propositus #2: A102/B101/O01. A panel of nine short-tandem repeat (STR) loci was tested on DNA extracted from blood, buccal mucosal cells, and hair from the propositi and on DNA isolated from their parents' blood. In all tissues tested from propositus #1, three loci demonstrated a double paternal and a single maternal DNA contribution, indicating that he was a chimera or a mosaic; in those from propositus # 2, one STR locus demonstrated a double paternal DNA contribution, indicating that he was a tetragametic chimera. Chimerism and mosaicism are uncommon but important causes of ABO genotype and phenotype discrepancies. The evaluation of patients and donors with unusual or unexpected serology in pretransfusion testing and consensus ABO alleles may include the evaluation of STR loci to detect these phenomena.

The genetic and phenotypic basis of blood group A subtypes in Koreans.

A serological and genetic study of Korean blood donors with phenotypic group A subtypes was performed. There were 176 donors with phenotypic A subtypes identified. Exons 6 and 7 from 57 representative donors were sequenced. The A(var) allele (784 G > A) was cloned and sequenced, and a family study demonstrating its inheritance and unusual serological characteristics was performed. The A102 allele was the most frequently identified allele in phenotypically A2 (58%, 11/19) and A2B (68%, 17/25) donors. Anti-A1 was rarely present amongst A2 and A2B donors. The family study revealed that the A(var) allele was expressed as phenotype A(weak)B in A(var)/B heterozygote members, but as phenotype O in A(var)/O heterozygotes. The most frequent allele in Korean donors with the A2 phenotype differs from its Caucasian counterpart, as does the frequency of anti-A1. The A(var) allele demonstrates allelic enhancement in A(var)/B heterozygotes.

A novel B(var) allele (547 G>A) demonstrates differential expression depending on the co-inherited ABO allele.

BACKGROUND AND OBJECTIVES: Genetic analysis of group B donors in Korea was performed. MATERIALS AND METHODS: Exons 6 and 7 were sequenced in 12 phenotypically B3 donors 6 B3, 6 A1B3. RESULTS: Consensus sequences all B3 and 2/6 A1B3 donors were present. Four A1B3 donors demonstrated a novel B allele, B(var), in the context of A101/ or A102/B(var) genotypes. Family studies based on an A1B3 donor with the B(var) allele and on another unrelated subject with identical genotype and phenotype revealed B(var)/O01 genotypes with full B-antigen expression. CONCLUSIONS: B(var) allele is subject to differential expression, depending on the co-inherited ABO allele.

The serological and genetic basis of the cis-AB blood group in Korea.

BACKGROUND AND OBJECTIVES: The cis-AB blood group is rare, although relatively common amongst Koreans. The serological characteristics and genetic basis of Korean cis-AB blood donors were investigated. MATERIALS AND METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), designed to detect the cis-AB01 allele, was performed on 194 AB samples which demonstrated weak or unusual expression of either or both of the A or B antigens. RESULTS AND CONCLUSIONS: Sixty cis-AB01 donors were identified. cis-AB01/O01 or O02 were the most common genotypes (36/60) detected only in A(2)B(3) donors, and cis-AB01/B101 (nine of 60) was the least common genotype identified only in A(2)B donors. Surprisingly cis-AB01/A102 (15/60) was identified in a variety of phenotypes (A(1)B(3), A(1)B(x) or el, A(int)B(3)).

Electrophoretic karyotype analysis of sequential Candida parapsilosis isolates from patients with persistent or pecurrent fungemia.

We assessed the genetic relatedness of sequential isolates of Candida parapsilosis during persistent or recurrent fungemia and the effect of central venous catheter (CVC) removal. Serial isolates of C. parapsilosis were obtained from 17 patients with persistent or recurrent fungemia over periods of up to 5 months. Forty-eight C. parapsilosis isolates from the blood of 17 patients were analyzed by electrophoretic karyotyping (EK) with pulsed-field gel electrophoresis (PFGE), revealing 25 different karyotypes. The strains sequentially isolated from each of seven patients whose fungemia resolved following CVC removal had the same karyotype. Two patients with fungemia that cleared without CVC removal each had two sequential isolates with different karyotypes. In six (75%) of the eight patients whose fungemia was recurrent even after CVC removal, the karyotypes of the pre- and post-CVC removal isolates were different, implying the emergence of a new strain. Overall, the sequential strains from each patient had identical karyotypes in 53% (9 of 17) of the patients and two different karyotypes in 47% (8 of 17). This study shows that EK with PFGE is useful for investigating persistent or recurrent fungemia due to C. parapsilosis and that recurrent fungemia due to C. parapsilosis is more likely caused by reinfection with a second strain.

Multiplex in-cell reverse transcription-polymerase chain reaction for the simultaneous detection of p210 and p190 BCR-ABL mRNAs in chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia cell lines.

We designed a novel multiplex in-cell reverse transcription-polymerase chain reaction method for the simultaneous detection and differentiation of p190 and p210 BCR-ABL mRNAs within single cells from the human chronic myeloid leukemia and Philadelphia positive acute lymphoblastic leukemia. Human K562 chronic myeloid leukemia and SUP B-15 Ph+ acute lymphoblastic leukemia cell lines were used as positive controls for p210 and p190 BCR-ABL mRNAs, respectively. HL60 cell line was used as a negative control. After the leukemia cells were fixed and permeabilized, without extracting nucleic acids, the mRNAs were reverse transcribed to cDNAs, and the cDNAs were amplified by multiplex polymerase chain reaction with fluorescent primers specific for p190 and p210 BCR-ABL mRNAs. After transfer onto glass slides by cytospin, the amplified cells were detected by fluorescence microscopy. Fluorescence microscopy after propidium iodide or 4',6-diamidino-2-phenylindone counterstaining showed that the positive K562 cells exhibited a yellow-green fluorescent cytoplasm around a red nucleus, and that the positive SUP B-15 cells exhibited an orange cytoplasm around a blue nucleus. Only the red or blue nucleus was visible in respective negative HL60 cells. The specificity of amplification was confirmed by the absence of a signal when control experiments were performed either with RNase digestion of mRNA or without reverse transcriptase/Taq polymerase. We conclude that the multiplex in-cell reverse transcription-polymerase chain reaction method is capable of simultaneously detecting and differentiating the p210 and p190 BCR-ABL mRNAs of chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia cells, and that it may be useful in quantitatively monitoring the minimal residual disease during therapy.

Nosocomial cluster of Candida lipolytica fungemia in pediatric patients.

Candida lipolytica has rarely been reported as a human pathogen. An apparent outbreak of Candida lipolytica fungemia (n = 5 cases) occurred in a pediatric ward over a 9-week period. The five patients infected were hospitalized in three adjacent rooms and cared for by the same healthcare workers. The index patient had central venous catheter-related fungemia, whereas the second patient, who was in the adjacent single room, had transient fungemia. Three additional cases of fungemia occurred in patients with hematological disorders who shared the same room; all three patients had central venous catheters and had been receiving oral fluconazole prophylaxis (50 mg/day for more than 3 weeks) at the time of infection. In vitro susceptibility testing of the strains showed that the MIC of fluconazole for all the isolates was 32 microg/ml. Random amplified polymorphic DNA analysis provided evidence of the clonal origin of the isolates, but the source of the outbreak was not identified. All four patients with persistent fungemia were successfully treated via catheter removal or empiric amphotericin B treatment. This outbreak shows the potential for the nosocomial epidemic transmission of Candida lipolytica.

Genetic variation of the Apo Al-CIII-AIV gene cluster in hypertriglyceridemic patients with chronic renal failure undergoing hemodialysis.

Many patients with chronic renal failure (CRF) requiring hemodialysis present with hypertriglyceridemia (HTG). But the exact cause of HTG in CRF is still unknown. Genetic variation of the apo AI-CIII-AIV gene cluster was reported to be associated with primary HTG, atherosclerosis and coronary artery disease. This study was designed to evaluate the association between the restriction fragment length polymorphism (RFLP) of the apo AI-CIII-AIV gene cluster and HTG in patients with CRF undergoing hemodialysis. Genetic variations of the apo AI-CIII-AIV gene cluster were analysed in peripheral leukocyte samples from 59 patients with CRF undergoing hemodialysis: 17 patients with HTG (CRF-HTG) and 42 patients without HTG (CRF-NTG). The RFLP was achieved through the digestion of PCR products by two restriction enzymes, SstI and MspI. The frequency of SstI minor allele (S2) in CRF-HTG was 0.44, which was significantly higher than that in CRF-NTG (0.17). Frequencies of MspI minor allele (M2) in CRF-HTG and CRF-NTG were not significantly different (0.5 vs 0.32) (p=0.07). Frequencies of S2-M2 genotype were 0.65 in CRF-HTG, and 0.27 in CRF-NTG (p<0.005). These data indicate that genetic variation of the apo AI-CIII-AIV gene cluster may serve as one of the causes of HTG in CRF.

Erythropoietin does not affect nitric oxide system in rats with chronic renal failure.

We investigated to see whether an altered role of nitric oxide (NO) system is involved in erythropoietin (EPO)-induced hypertension in chronic renal failure (CRF). Male Sprague-Dawley rats were five-sixths nephrectomized to induce CRF. Six weeks after the operation, EPO or vehicle was injected for another 6 weeks. Plasma and urine nitrite/nitrate (NOx) levels were determined. Expression of NO synthase (NOS) proteins in the aortae and kidneys were also determined. In addition, the isometric tension of isolated aorta in response to acetylcholine and nitroprusside was examined. Blood pressure progressively rose in CRF groups, the degree of which was augmented by EPO treatment. Plasma NOx levels did not differ among the groups, while urine NOx levels were lower in CRF groups. Endothelial NOS expression was lower in the kidney and aorta in CRF rats, which was not further affected by EPO-treatment. The inducible NOS expression in the kidney and aorta was not different among the groups. Acetylcholine and sodium nitroprusside caused dose-dependent relaxations of aortic rings, the degree of which was not altered by EPO-treatment. Taken together, EPO-treatment aggravates hypertension in CRF, but altered role of NO system may not be involved.

Molecular typing of vibrio vulnificus isolated from clinical specimens by pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis.

Primary V. vulnificus septicemia has been continuously reported in Korea. We analyzed the molecular diversity of V. vulnificus strains isolated from clinical specimens using pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. We analyzed a total of 23 V. vulnificus strains isolated from 22 patients between 1992 and 1997. RAPD analysis was performed using five random primers, and we obtained chromosomal DNA restriction patterns with NotI and SfiI by PFGE. Two isolates from one patient disclosed similarity value 1.0 by RAPD and had an indistinguishable pattern when analyzed with PFGE, which indicated that they were the same strains. The remaining 22 isolates from 22 patients could be separated into 5 different molecular types in RAPD analysis. The range of similarity values among the isolates was wide (0.29-0.81). Of 22 strains, four strains could not be typed in repeated trials by PFGE with NotI and SfiI. The PFGE patterns of 18 strains showed a remarkable polymorphism consisting 12-19 fragments (20-870 kb). These results show that V. vulnificus strains isolated from Korea are genetically diversified for the most part.

Fatal Hormonema dematioides peritonitis in a patient on continuous ambulatory peritoneal dialysis: criteria for organism identification and review of other known fungal etiologic agents.

We report a fatal case a fungal peritonitis caused by the yeast-like dematiaceous mould Hormonema dematioides in a 45-year-old woman. The woman had a 13-year history of insulin-dependent diabetes mellitus and had been on continuous ambulatory peritoneal dialysis for chronic renal failure. H. dematioides was repeatedly isolated from the dialysate culture specimens collected on days 3, 9, 16, and 20 of her hospital stay. Preliminary culture reports on day 7 of the growth of a yeast-like fungus, a probable Candida species, prompted the administration of fluconazole (FLU). Intraperitoneal and intravenous FLU failed to eliminate the mould, and the patient expired on day 21 of her hospital stay. We use this case to present what appears to be the first report of fungal peritonitis due to H. dematioides, to provide laboratorians with criteria for differentiating this organism from the similar mould Aureobasidium pullulans and from various yeast genera, and to provide a review of known fungal taxa inciting peritonitis.

Molecular epidemiologic analysis of Staphylococcus aureus isolated from clinical specimens.

Nosocomial infections caused by Staphylococcus aureus are clinically serious and control of such infections requires strain typing to identify the source of contamination. Recently, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) assay have been introduced and have provided a high level of strain discrimination of S. aureus isolated from clinical specimens. This study was performed to classify 82 strains of S. aureus isolated from 4 hospitals in the Kwangju-Chonnam area by PFGE and RAPD assay. Methicillin-resistant S. aureus (MRSA) was identified by disk diffusion method using the oxacillin disk and polymerase chain reaction of mecA gene was done in 69 strains. Eight-three strains including S. aureus ATCC 25923 were classified into 10 groups by RAPD assay, and into 8 groups by PFGE. Classified groups were not related to area or hospital. Classification was not characteristic between MRSA and methicillin-susceptible strains. Nosocomial infections due to outbreak were suggested because some strains disclosed identical band patterns by PFGE. These results indicate that medical personnels and instruments are routes of nosocomial infections caused by MRSA. PFGE and RAPD assay are powerful tools for the epidemiological study of S. aureus, but PFGE is more effective than RAPD assay. RAPD assay needs optimal combination of primers.

Aspergillus nidulans infection in a patient with chronic granulomatous disease.

Aspergillus nidulans is one of the several species of Aspergillus with low pathogenicity. The significant infections of A. nidulans in human have rarely been reported, almost exclusively in patients with chronic granulomatous disease (CGD). CGD is a primary immunodeficiency disease which results from the absence of the NADPH oxidase in the phagocytic cells, leading to recurrent pyogenic infection and granuloma and abscess formation. Here we report a fatal case A. nidulans infection in a six-year-old boy with chronic granulomatous disease. A. nidulans was isolated from the culture of a paraspinal abscess and Aspergillus was detected in the surgical tissue by in situ hybridization. The patient succumbed despite prolonged treatment with high-dose amphotericin B, itraconazole and interferon-alpha. To our knowledge, this is the first report of A. nidulans infection in Korea.

Molecular typing of Vibrio vulnificus isolates by random amplified polymorphic DNA (RAPD) analysis.

This study was undertaken to determine molecular types and genetic similarity among V. vulnificus isolates by RAPD analysis. We compared these results with serotypes of V. vulnificus. Ninety-seven V. vulnificus strains including 69 strains from Chonnam University Hospital (CUH; Kwangju, Korea), 13 from Wonkwang University Hospital (WUH; Iksan, Korea), 13 from the Japanese National Institute of Health (JNIH) and two reference strains (ATCC 33815 and ATCC 27562) were analyzed. Four molecular types comprising all the strains were obtained by RAPD analysis. Type I was the most common (60/95) and included 58 strains from CUH. Type I showed a further subdivision into seven subtypes. Type II (23/95) composed of 11 strains from CUH, nine from WUH, three from JNIH and two reference strains. Six type III strains comprised four WUH strains and two JNIH strains. All six strains of type IV were from JNIH. The range of genetic similarity values among V. vulnificus isolates was 0.24 to 1.00. The serotypes of 95 strains were 04 (84.2%), 014 (3.2%), 01 (2.1%), 013 (2.1%), and R (2.1%). The most common 04 serotype strains were distributed among types I (60 strains), II (23 strains), III and IV (six strains). Although the V. vulnificus isolates showed a wide range of genetic similarity values, RAPD analysis could separate V. vulnificus strains into four molecular types, and the isolates from the same hospitals tended to belong to the same molecular type. There was no specific correlation between molecular types and serotypes of V. vulnificus.

Alternative use of polymerase chain reaction instead of rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone test for the early detection of Mycobacterium tuberculosis in BACTEC 12B cultures.

Compared with conventional culture media, the TB BACTEC system has demonstrated improved isolation rates as well as an earlier detection time for mycobacterial species. However, the identification of M. tuberculosis by the rho-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test in the TB BACTEC 460 system may require 6 days for interpretable results. We evaluated the usefulness of a polymerase chain reaction (PCR) assay for earlier identification of M. tuberculosis in positive BACTEC 12B cultures. A total of 262 TB BACTEC culture specimens with GIs > or = 10 were assayed by PCR, and the results were compared with those of the NAP test. The aliquot from BACTEC 12B vials was boiled for 10 min, and 2 microliters of the boiled suspension was used for the PCR assay. One set of primers based on the IS 6110 sequence of M. tuberculosis was used to amplify a 457 bp fragment of DNA. Of the 173 TB BACTEC culture specimens which were identified as M. tuberculosis by the NAP test. 171 were PCR positive. Of the 21 TB BACTEC cultures identified as MOTT by the NAP test. 19 were PCR negative, but 2 were PCR positive: these two cultures were shown to grow both M. tuberculosis and MOTT in BACTEC 12B vials. Of the remaining 68 cultures which were contaminated with AFB-negative bacteria, the PCR identified M. tuberculosis in 13, in agreement with the NAP results in the reprocessed specimens. Overall, the PCR results in the 262 BACTEC culture specimens with GIs > or = 10 were sensitive in 99.5% (186/187) and specific in 100% (68/68). The mean time for the identification of M. tuberculosis in TB BACTEC cultures with GIs > or = 10 was 7 h by the PCR compared to 5.9 days by the NAP test. These results suggest that the PCR could be used as an alternative to the NAP test for the rapid identification of M. tuberculosis in BACTEC 12B cultures, particularly in those which contained both M. tuberculosis and MOTT or M. tuberculosis and AFB-negative bacteria.