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)).

Actinomycin D renders cultured synovial fibroblasts susceptible to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis.

OBJECTIVE: To investigate the expression of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors in cultured synovial fibroblasts from rheumatoid arthritis (RA) and osteoarthritis (OA) patients, and to examine their susceptibility to TRAIL-induced apoptosis in the presence or absence of metabolic inhibitors. METHODS: The expression of TRAIL receptors in synovial fibroblasts was examined by Western blot and immunohistochemistry. Expression of TRAIL-receptor 1 (TRAIL-R1), FLICE-inhibitory protein (Fas-associating protein with death domain-like interleukin-1-converting enzyme), and Bcl-2 was assessed by Western blot. Synovial cell viability was measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay (XTT), and apoptosis was determined both by DNA content analysis after propidium iodide staining and Annexin V stain. RESULTS: TRAIL-R1 was constitutively expressed on cultured synovial fibroblasts from RA and OA, however, expression of TRAIL-R2 and TRAIL-R3 was not observed by immunohistochemistry and Western blot. Cultured synovial fibroblasts were resistant to apoptosis by TRAIL alone, but combined treatment of TRAIL with actinomycin D (ActD: 200 ng/mL), cycloheximide (CHX: 10 microg/mL), or proteasome inhibitor (MG132: 20 microM) induced apoptosis in a dose-dependent manner. The apoptosis was completely or partially inhibited by various caspase inhibitors, implicating an involvement of caspase pathway in TRAIL-induced apoptosis in the presence of these metabolic inhibitors. Expression of TRAIL-R1, FLIPL, and Bcl-2 did not account for the apoptosis by the combined treatment of TRAIL with ActD. CONCLUSIONS: Although TRAIL-R1 was constitutively expressed; cultured synovial fibroblasts were resistant to apoptosis by TRAIL. ActD, CHX, and MG132 rendered cultured synovial fibroblasts susceptible to TRAIL-induced apoptosis by a caspase-dependent mechanism. However, the exact mechanism of sensitization by these metabolic inhibitors remains to be determined.

Increased angiogenesis and Fas-ligand expression are independent processes in acute myeloid leukemia.

We evaluated the clinical significance of tumor angiogenesis and Fas-ligand (FasL) expression using parameters including the microvessel count (MVC), vascular endothelial growth factor (VEGF) level, and FasL expression in patients with acute myeloid leukemia (AML). Paraffin-embedded bone marrow (BM) sections from 43 AML patients at diagnosis, 20 patients after subsequent induction therapy, and 18 controls with non-invasive lymphoma were stained immunohistochemically for von Willebrand factor (vWF) and FasL. VEGF in BM mononuclear cells from 32 AML patients at diagnosis and 10 controls, including bone marrow transplantation donors, was assayed by an ELISA method. We found that the mean MVC, VEGF level, and FasL expression in AML patients at diagnosis were significantly higher than those of controls, with a significant correlation between the MVC and VEGF levels (r=0.43). However, there were no correlations between FasL expression and MVC or VEGF level. The mean MVC and FasL expression after induction therapy were lower than those evaluated at diagnosis, but were higher than those of controls. There was a correlation between the MVC and percentage of BM blasts (r=0.479), but no correlation between the MVC, VEGF level, or FasL expression and other hematologic or clinical variables. Our findings provide evidence of increased angiogenesis and tumor immune escape in AML, and both angiogenesis and tumor immune escape are independent processes in AML.

Secondary myeloid/natural killer cell acute leukemia following T-cell lymphoma.

A 56-year-old woman was treated with combination chemotherapy and radiation therapy for peripheral T-cell lymphoma. Following complete remission for a period of 6 months, she returned again with marked leukocytosis. Leukemic cells were characterized by scanty cytoplasm with fine azurophilic granules, and were highly positive for myeloperoxidase and sudan black-B. Immunophenotypic analysis revealed that blast cells were positive for myeloid antigens (CD13, CD33), and natural killer (NK) cell antigen (CD56), but negative for T-cell antigens (CD2, CD5, CD7), B-cell antigens (CD19, CD20), CD34, and HLA-DR. The case was diagnosed as secondary myeloid/NK cell acute leukemia following non-Hodgkin's lymphoma. Despite aggressive chemotherapy against leukemia, she died of multiorgan failure 7 months following onset of leukemia. We present, to the best of our knowledge, the first published report of what seems to be a secondary myeloid/NK cell acute leukemia following T-cell lymphoma.

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.

Clinical efficacy of granulocyte transfusion therapy in patients with neutropenia-related infections.

Granulocyte transfusions have been advocated by some for the treatment of severe, progressive infections in neutropenic patients who fail to respond to antimicrobial agents and recombinant hematopoietic growth factors. We conducted the current study to determine an appropriate method of granulocyte mobilization in healthy donors, and to evaluate the safety and efficacy of granulocyte transfusion therapy in patients with neutropenia-related infections. To mobilize granulocytes (n=55), healthy normal donors were stimulated in one of the following ways: (1) dexamethasone, 3 mg/m2 intravenously 15 min prior to leukapheresis (n = 5); (2) granulocyte colony-stimulating factor (G-CSF), 5 microg/kg subcutaneously 12 to 14 h prior to collection (n=37); or (3) G-CSF and dexamethasone (n= 13). The mean granulocyte yield from stimulation with G-CSF plus dexamethasone was significantly higher than from stimulation with dexamethasone or G-CSF alone. Twenty-five patients with severe neutropenia-related infections unresponsive to appropriate antimicrobial agents received a total of 55 granulocyte transfusions. The patients from whom fungi or Gram-negative organisms were isolated showed a more favorable response than those infected with Gram-positive organisms. However, the responses to the granulocyte transfusion therapy could not be correlated with the transfused dose, mobilization agents, or the 1 h or 24 h post-transfusion absolute neutrophil counts. We conclude that granulocyte transfusion therapy may be clinically useful for neutropenia-related infections by fungi or Gram-negative organisms.

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.

Acquired hemophilia successfully treated with oral immunosuppressive therapy.

Acquired hemophilia is a rare disorder associated with serious bleeding in nonhemophilic patients. We had a 40-year-old woman who was diagnosed as acquired hemophilia with a factor VIII inhibitor level of 27.5 BU/mL. She was presented with gross hematuria and severe right shoulder pain, and was successfully treated with daily oral cyclophosphamide and prednisone for 2 weeks. After the remission, the doses of prednisone and cyclophosphamide were slowly decreased and she remained in complete remission without further bleeding episodes.

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.

Successful allogeneic bone marrow transplantation for childhood-onset refractory anemia with ringed sideroblasts.

Refractory anemia with ringed sideroblasts (RARS) is an extremely rare type of myelodysplastic syndrome in children. We describe a 10-year-old boy with RARS presented with pancytopenia. He remained relatively stable with only a few transfusions until age of 20 years, when he underwent an allogeneic bone marrow transplantation (BMT) because of increased transfusion requirements. He remains in complete chimeric state at 20 months posttransplant with normal hematologic parameters. To our knowledge, this is the first description of successful BMT in a patient with childhood-onset RARS. The indication of BMT for this rare disorder in children is discussed.

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.

Acute hemolytic crisis with fulminant hepatic failure as the first manifestation of Wilson's disease: a case report.

We report a 27-year-old woman who developed Coombs' negative hemolytic anemia and fulminant hepatic failure as the initial manifestation of Wilson's disease. Unmeasurably low level of serum alkaline phosphatase provided a clue to the diagnosis of Wilson's disease. The diagnosis was established with the presence of Kayser-Fleischer ring, decreased serum ceruloplasmin level, and elevated urine and serum copper levels. In spite of repeated plasmapheresis, she died of multiorgan failure on the fifth hospital day.