N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

Evaluation of the AutoLab HbA1c Reagent by Using Hitachi Clinical Analyzer 7180

BACKGROUND: Hemoglobin A1c (HbA1c) is a universally used parameter for monitoring glycemic control in diabetic patients. Various methods are used for the measurement of HbA1c levels. The AutoLab HbA1c reagent was recently developed for use in immunoturbidimetric assays for measurement of HbA1c levels. We evaluated the reliability of using the AutoLab HbA1c reagent with the Hitachi Clinical Analyzer 7180, an automated chemistry analyzer, for measuring HbA1c levels. METHODS: We evaluated the precision, linearity, carryover rate, and stability of the samples analyzed using Hitachi clinical analyzer 7180 with the AutoLab HbA1c reagent and compared the results with those obtained with an HPLC method performed using the Variant II Turbo analyzer. RESULTS: The CV values for within-run imprecision at low and high levels were 0.8% and 0.6%, respectively, and the CV values for between-run imprecision at low and high levels were 1.5% and 2.9%, respectively. The linearity of the results was good in the range of 4.3-12.3%, and comparison with the results obtained by Variant II Turbo showed an excellent correlation coefficient of 0.9914. The carryover rate was 0%, and the samples refrigerated at 4degrees C for 15 days were found to be stable. CONCLUSIONS: In comparison with Variant II Turbo, Hitachi clinical analyzer 7180 with AutoLab HbA1c reagent showed good precision, linearity, and carryover rate. Hence, Hitachi clinical analyzer 7180 with AutoLab HbA1c reagent may be used for the diagnosis of diabetes and for monitoring blood glucose levels in diabetic patients.

A case of therapy-related acute myeloid leukemia with inv(16)(p13.1q22) after single low-dose iodine-131 treatment for thyroid cancer / 대한혈액학회지

Radioiodine is regularly used in the treatment of thyroid cancer to eliminate residual malignant tissue after thyroidectomy and to treat metastasis. Because of the low dose of radioiodine used to treat thyroid cancer patients, leukemia is an uncommon complication of exposure to radioiodine. Here, we present a patient who developed therapy-related acute myeloid leukemia with inv(16)(p13.1q22);CBFbeta-MYH11, eosinophilia, and K-ras mutation and who had been treated with very low-dose radioiodine following total thyroidectomy.

A Case of Immune Hemolytic Anemia due to Autoantibodies Against C and e Antigens in a Patient with Paroxysmal Nocturnal Hemoglobinuria and Myelodysplastic Syndrome / 대한수혈학회지

Antiglobulin test-negative hemolytic anemia, thrombophilia, and marrow failure, such as aplastic anemia and myelodysplastic syndrome - refractory anemia (MDS-RA), are the primary clinical manifestations of paroxysmal nocturnal hemoglobinuria (PNH). Here, we report on a case of a 56-year-old male patient diagnosed with PNH, MDS-RA, and immune hemolytic anemia (IHA). The patient was transferred to the hospital with an impression of hemolytic anemia and pulmonary embolism. Positive results were observed on direct and indirect antiglobulin tests, and alloantibody, anti-C and anti-e, autoantibodies were identified. In addition, C and e antigens were found in Rh subgrouping. Therefore, due to the presence of autoantibodies against C and e antigens, we assumed that the cause of IHA was autoimmune reaction. Spherocytosis, increased osmotic fragility test, and positivity on direct and indirect antiglobulin tests were not considered characteristics of PNH. Therefore, without the presence of pulmonary embolism and MDS-RA, it is possible that autoimmune hemolytic anemia was considered the only reason for the hemolytic anemia, and that PNH could be overlooked. In patients with PH, use of washed RBCs during transfusion is not necessary. PNH screening test is recommended for patients who have experienced a thromboembolic event and intravascular hemolysis or MDS-RA. In order to obtain accurate information regarding the percentage of GPI-AP-deficient RBCs, flow cytometric analysis should be performed prior to transfusion.

A Case of B-cell Lymphoma, Unclassifiable, with Features Intermediate between Diffuse Large B-cell Lymphoma and Burkitt Lymphoma in a Korean Child

B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) (intermediate DLBCL/BL), is a heterogeneous group with some features resembling DLBCL and others resembling BL. Here, we report a case of intermediate DLBCL/BL in a Korean child. A 2-yr-old male was admitted for evaluation and management of left hip pain. Immunohistochemistry of a biopsy of the femur neck revealed tumor cells positive for CD20, CD10, BCL2, BCL6, and Ki67. A bone marrow (BM) aspirate smear revealed that 49.3% of all nucleated cells were abnormal lymphoid cells, composed of large- and medium-sized cells. Immunophenotyping of the neoplastic cells revealed positivity for CD19, CD10, CD20, and sIg lambda and negativity for CD34, Tdt, and myeloperoxidase (MPO). Cytogenetic and FISH analyses showed a complex karyotype, including t(8;14)(q24.1;q32) and IGH-MYC fusion. Intensive chemotherapy was initiated, including prednisone, vincristine, L-asparaginase, daunorubicin, and central nervous system prophylaxis with intrathecal methotrexate (MTX) and cytarabine. One month after the initial diagnosis, BM examination revealed the persistent of abnormal lymphoid cells; cerebrospinal fluid cytology, including cytospin, showed atypical lymphoid cells. The patient was treated again with cyclophosphamide, vincristine, prednisone, adriamycin, MTX, and intrathecal MTX and cytarabine. The patient died of sepsis 5 months after the second round of chemotherapy.

A Case of Scedosporium Apiospermum Pneumonia in an Immunocompetent Patient / 대한내과학회지

Scedosporium apiospermum, an asexual form of Pseudallescheria boydii, is a saprophytic mold with a worldwide distribution. It may cause severe pulmonary or disseminated infections in immunocompromised patients who have undergone organ transplantation, have hematological malignancies, or have received corticosteroid therapy. However, in immunocompetent patients, it usually produces localized infection and has been reported to cause pneumonia after near-drowning in polluted water. We present here the case of an immunocompetent 72-year-old woman with pneumonia caused by S. apiospermum.

Reticulocyte Indices for the Differential Diagnosis of Anemia of Chronic Disease and Iron Deficiency Anemia in Adult Women with Microcytic Hypochromic Anemia

BACKGROUND: Iron deficiency anemia (IDA) is the most common anemia followed by anemia of chronic disease (ACD). Reticulocyte indices have been shown to be helpful indicators for detecting IDA. We investigated whether RBC and reticulocyte indices can be used to differentiate ACD from IDA. METHODS: A total of 85 women showing microcytic hypochromic anemia (38 IDA and 47 ACD cases) were enrolled. IDA was defined as those with ferritin level of 450 microg/dL. ACD was defined as ferritin level of > or =6 microg/dL, TIBC of or =24.6 pg could be used to differentiate ACD from IDA with 85.1% sensitivity and 81.6% specificity. CONCLUSIONS: The reticulocyte indices, especially CHr, are useful for the differential diagnosis of microcytic hypochromic anemias, ACD and IDA.

Characteristics of CTX-M Type Extended Spectrum beta-lactamase Producing Non-typhoi dal Salmonella Isolates

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing Salmonella have been increasingly reported worldwide. ESBL-producing Salmonella is of particular concern since children cannot be treated with quinolones. This study was conducted to determine the phenotypic and genetic characteristics of ESBL-producing Salmonella in a tertiary hospital. MATERIALS AND METHODS: Four clinical ESBL-producing isolates of non-typhoidal Salmonella were collected during 2001 to 2009. Antimicrobial susceptibility was determined by disk diffusion test and VITEK-II system. ESBL production was tested by ESBL phenotypic confirmatory test. TEM, SHV, CTX-M1, CTX-M2, CTX-M8, and CTX-M9 type ESBL genes were detected by PCR amplification, and PCR products were subjected to direct sequencing. RESULTS: Phenotypic confirmatory test showed that 4 of the 300 non-typhoidal Salmonella isolates were ESBL-producing: 3 S. Enteritidis and 1 S. Typhimurium. All 4 isolates were recovered during the past 1 year period. All 3 S. Enteritidis harbored CTX-M-15, while the S. Typhimurium harbored CTX-M-14. All CTX-M-15-producing S. Enteritidis isolates showed resistance both to cefotaxime and ceftazidime, while the CTX-M-14-producing S. Enteritidis were resistant only to cefotaxime. CONCLUSIONS: ESBL-producing nontyphoidal Salmonella has emerged recently and the type of ESBL has switched from TEM and SHV to CTX-M.

Prevalence of Inducible Macrolide-Lincosamide-Streptogramin B (MLS(B)) Resistance in Erythromycin-Resistant Staphylococci

BACKGROUND: Inducible MLS(B) (macrolide-lincosamide-streptogramin B) resistance in staphylococci is not detected by standard susceptibility test methods. Failure to identify inducible MLS(B) resistance may lead to clinical failure during clindamycin therapy. We determined the prevalence of inducible MLS(B) resistance in erythromycin-resistant staphylococcal isolates. MATERIALS AND METHODS: We evaluated all 2,792 non-duplicate staphylococcal strains: 1,402 Staphylococcus aureus and 1,390 coagulase-negative staphylococci (CoNS) isolated from May 2008-June 2009 at one-unoversity hospital. Testing for inducible MLS(B) was accomplished by the disk approximation test (D-test) in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI). RESULTS: Of the 2,792 staphylococcal isolates, 892 S. aureus isolates and 740 CoNS isolates were resistant to erythromycin. Among the 892 erythromycin-resistant S. aureus isolates, the overall prevalence of inducible MLS(B) was 21.3% (16.2% of MRSA and 76.3% of methicillin-susceptible S. aureus). Among the 740 erythromycin-resistant CoNS isolates, the overall prevalence of inducible MLS(B) was 16.5% (16.0% of methicillin-resistant CoNS and 18.7% of methicillin-susceptible CoNS). The D-test was positive in 88.8% of S. aureus and 28.4% of CoNS isolates, which were erythromycin-resistant and clindamycin-susceptible. CONCLUSIONS: There are some variations in the prevalence of inducible MLS(B) resistance in clinical staphylococcal isolates. It is important that clinical laboratories report inducible MLS(B) resistance for erythromycin-resistant and clindamycinsusceptible staphylococcal isolates.

Impact of Revised Penicillin Breakpoints for Streptococcus pneumoniae (CLSI M100-S18) on the Penicillin Susceptibility Rate / 대한임상미생물학회지

BACKGROUND: In January 2008, the Clinical and Laboratory Standards Institute (CLSI) published revised penicillin breakpoints for Streptococcus pneumoniae according to clinical presentation and the route of penicillin administration. The aim of this study was to evaluate the impacts of the new penicillin breakpoints on the susceptibility rates of S. pneumoniae isolated from blood. METHODS: A total of 156 non-duplicated S. pneumoniae strains recovered from blood of hospitalized patients were collected between January 2003 and December 2008. Penicillin and cefotaxime susceptibility tests were performed using an E-test (AB Biodisk, Solna, Sweden). Results of the penicillin susceptibility tests were analyzed using the former and new CLSI guidelines. RESULTS: Of the 156 S. pneumoniae strains isolated from blood, penicillin susceptibility under the former CLSI guidelines resulted in 42.3% susceptible, 42.3% intermediate, and 15.4% resistant states. According to the new CLSI guidelines (nonmeningitis, parenteral), 87.8% of isolates were susceptible, 9.6% were intermediate, and 2.6% were resistant to penicillin. CONCLUSION: When the new CLSI guidelines are applied, the penicillin susceptibility rate of S. pneumoniae strains isolated from blood is considerably increased. This suggests that penicillin should still be useful for the treatment of nonmeningeal pneumococcal infections and that the use of broad-spectrum antimicrobials should not replace this treatment.

A Case of Non-secretory Myeloma with Crystal-storing Histiocytosis / 대한진단검사의학회지

Crystal-storing histiocytosis (CSH) is a rare event observed in association with lymphoproliferative diseases, and mainly occurrs in plasma cell dyscrasias. It is presumed to be an intra-lysosomal accumulation of the secreted paraproteins. Crystal formation can be seen inside histiocyte-like cells with phagocytosed crystalline inclusions in the bone marrow and extramedullary sites. CSH is a rare morphological entity with poor prognostic implications and may be confused with Gaucher or pseudo-Gaucher cells. Herein we report a case of non-secretory myeloma associated with CSH showing a poor clinical course. A 79-yr-old male presenting with dizziness was evaluated in hematology department for anemia. Laboratory tests revealed Hb of 4.9 g/dL and beta2-microglobulin of 21,000 ng/mL (reference range, 0-370). Presence of monoclonal protein was not detected on protein electrophoresis and immunofixation in serum and urine. However, serum free light chain assay showed an increased kappa-light chain level of 126 mg/L (reference range, 3.3-19.4) resulting in an increased kappa/lambda ratio. The bone marrow touch print showed numerous plasma cells and crystal-laden histiocytes and immunohistochemical stainings on bone marrow biopsy revealed positivity for CD38, CD56 and kappa in the plasma cells and CD68 and kappa in crystal-laden histiocytes.

t(9;22) with 5'ABL1 Deletion and t(6;19) in Biphenotypic Acute Leukemia / 대한혈액학회지

Biphenotypic acute leukemia (BAL) is a rare type of leukemia, comprises 4% of all acute leukemias. It is more common in adults and the clinical features, as related to marrow dysfunction, are similar to those found in other patients with acute leukemia. BAL commonly shows a dimorphic blast population with, one resembling lymphoblasts and the other resembling myeloblasts. The majority of BAL patients express B-lymphoid and myeloid markers. BAL can be diagnosed by morphologic studies and by a comprehensive panel of immunological markers, as well as cytogenetic/molecular studies, such as fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, its prognosis is relatively poor. We present here a 27 year-old female patient who showed lymphoblasts and myeloblasts on her marrow studies and these cells were positive for myeloid and B-lymphoid markers on the immunophenotypic studies. Chromosome analysis revealed 46,XX,t(6;19)(p23;p13.1),t(9;22)(q34;q11.2). A major (b3a2) type of BCR-ABL1 mRNA transcript was detected by RT-PCR, and a 5'ABL1 deletion was identified by FISH.

A Case of Primary Autoimmune Myelofibrosis / 대한혈액학회지

Myelofibrosis is usually observed in association with hematologic malignancies or metastatic solid tumors, but it has rarely been reported in patients who suffer with autoimmune disorders. Autoimmune myelofibrosis is a distinct clinicopathologic entity and it can occur alone or in association with autoimmune disorders, and the final result is chronic peripheral cytopenia. Primary autoimmune myelofibrosis, in which the autoimmune myelofibrosis is not preceded by a well-defined autoimmune disease, has recently been defined as a distinct clinicopathologic syndrome. We report here on a case of an 18-year-old woman who was diagnosed with primary autoimmune myelofibrosis, and she manifested peripheral pancytopenia, positivity for autoantibodies and Grade III myelofibrosis without having any preceding autoimmune or hematologic disorders.

Evaluation of Platelet Indices for Differential Diagnosis of Thrombocytosis by ADVIA 120 / 대한진단검사의학회지

BACKGROUND: For the diagnosis of essential thrombocythemia (ET), no single clinical or laboratory finding of diagnostic value is available and a differential diagnosis of other myeloproliferative neoplasms or reactive thrombocytosis (RT) is needed. Following recent developments in automated blood cell analyzers, various platelet indices can now be measured. In this study, we analyzed whether platelet counts and 6 platelet indices can be used for the differentiation of ET from RT in patients with a platelet count of 600x10(3)/microliter or more. METHODS: The subjects studied were 31 patients with ET and 224 patients with RT. The platelet counts, mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), mean platelet mass (MPM), mean platelet component concentration (MPC) and large platelets (LPLT) were measured by ADVIA 120 (Bayer Diagnostics, USA). The mean values of each item were compared between the two patient groups and the sensitivity and specificity of each item in the diagnosis of ET were determined by ROC curve analysis. RESULTS: In essential thrombocythemia, all parameters except MPC were significantly higher than in reactive thrombocytosis. For the diagnosis of ET, the sensitivity and specificity were: 74.2% and 84.4%, when the platelet count was > or = 820x10(3)/microliter; 80.6% and 80.0%, when the plateletcrit was > or =0.63%; and 64.5% and 99.1%, respectively, when LPLT was > or = 23x10(3)/microliter. CONCLUSIONS: The platelet counts and platelet indices are useful for the differential diagnosis of thrombocytosis. The plateletcrit and LPLT are particularly useful for the diagnosis of ET when the platelet count is markedly increased.

Peritonitis due to Leclercia adecarboxylata in a Patient Receiving Automated Peritoneal Dialysis / 대한신장학회지

Peritonitis in patients undergoing peritoneal dialysis is a major complication and the leading cause of peritoneal dialysis failure. Leclercia adecarboxylata is a motile, gram-negative, facultative anaerobic bacillus of the Enterobacteriaceae family. These bacteria are uncommon pathogen, and rarely isolated from environmental and clinical specimens. Some cases have been reported about peritonitis due to Leclercia adecarboxylata in a patient receiving continuous ambulatory peritoneal dialysis (CAPD). However, there has never been any report about peritonitis in a patient receiving automated peritoneal dialysis (APD). We have isolated Leclercia adecarboxylata from peritoneal fluid in a patient receiving APD, and the patient completely recovered with 14-day treatment of intraperitoneal antibiotics without catheter removal.