BACKGROUND: Dual esterase reaction enables simultaneous visualization of the cells exhibiting different characteristics by combining two esterase stains on a single slide. However, most methods previously reported require the smear slides to be stained twice, using separate coupling reagent with each substrate. Authors attempted to combine chloroacetate esterase (CAE) and alpha-naphthyl butyrate esterase (ANBE) staining methods by using single incubation and single mixed reagent. METHODS: For dual cytochemical reaction, buffy coat slides of normal subjects and bone marrow smear slides of patients with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) were used. Eighty mg of fast blue BB salt was dissolved in 50 mL of 0.1 mol/L phosphate buffer (pH 8.0). In the fast blue BB solution, 2 mL of acetone containing 4 mg of naphthol AS-D chloroacetate (Sigma, USA) and 8 mg of alpha-naphthyl butyrate (Sigma, USA) was added. Air dried slides were fixed with buffered formalin-acetone solution for 1 minutes. The fixed slides were allowed to be incubated with the single mixed reagent solution at room temperature for 20 minutes. Aquous hematoxylin dye was used for counterstaining. RESULTS: Granulocytes were stained bright blue by single-incubated dual esterase reaction. Monocytes and their precursors showed dark brownish positivity in cytoplasmic granules. Megakaryocytes in bone marrow revealed positive reaction for only ANBE by dual esterase method. The activity of CAE in myeloid lineage was markedly decreased in patients with MDS and some cells revealed dual staining activity with both punctate blue and brownish granules. A few neutrophils in karryorrhexis observed in remission marrow of AML patient showed strong positive reaction for CAE. CONCLUSIONS: Dual esterase staining method using single incubation is very simple in staining procedure and of use in simply identifying cellular characteristics and differentiation of myeloid and monocytic lineages on a single slide.
Acetone ; Bone Marrow ; Butyrates ; Coloring Agents ; Cytoplasmic Granules ; Granulocytes ; Hematoxylin ; Humans ; Leukemia, Myeloid, Acute ; Megakaryocytes ; Monocytes ; Myelodysplastic Syndromes ; Neutrophils

BACKGROUND: The purpose of this study is to investigate the prevalence of impaired platelet responsiveness to epinephrine in healthy subjects. Also, we compared the platelet aggregability in response to various agonists in normal population. METHODS: A total of 156 healthy subjects aged 21 to 57 years were investigated for the evidence of impaired responsiveness to epinephrine. Aggregometer PACKS-4 (Platelet Aggregation Chromogenic Kinetic System-4, Helena, Beaumont, USA) was used for platelet function test. Aggregating agonists (Helena Haemostasis Systems, UK) used in the study were consisted of ADP (10 micrometer), collagen (10 microgram/mL), epinephrine (300 micrometer) and ristocetin (1500 microgram/mL). Population showing platelet aggregability with more than 60% activity was classified as normal group, while aggregability with less than 20% as impaired responsiveness. RESULTS: Of 156 healthy subjects, 20.5% (32/156) showed impaired responsiveness, while 33.9% (53/156) revealed decreased aggregability with the activity of less than 60% to epinephrine. The mean of maximal percent aggregating activity for collagen was 90.5+/-11.4% and that of epinephrine was 66.5+/-34.4%. The mean aggregation activity (84.4+/-11.8%) for ADP in subjects showing normal response to epinephrine was significantly higher, compared with that (65.7+/-16.2%) of impaired responsiveness group to epinephrine (P<0.01). CONCLUSION: Impaired responsiveness to epinephrine, which is observed in healthy subjects, appears to be a kind of normal variant reaction. And this abnormality is not considered to be associated with any evident bleeding disorders.
Adenosine Diphosphate ; Blood Platelets* ; Collagen ; Epinephrine* ; Hemorrhage ; Platelet Aggregation ; Platelet Function Tests ; Prevalence* ; Ristocetin

Chronic myelogenous leukemia (CML) has been reported to show extra-medullary involvement in 10% of cases and affect mostly lymph nodes and spleen. Extensive pleura involvement of leukemic cells is very rare and no cases of CML infiltrating pleura have been reported in Koreans. Authors recently experienced a case of CML presenting pleural effusion with malignant leukemic cells. A 76-year-old woman was admitted at Inha University Hospital because of dyspnea and rectal bleeding 3 months ago. The patient had a past history of treatment for massive pleural effusion in local clinic 4 month ago. In biochemical test, LD (lactate dehydrogenase) was 620 IU/dL, and serum uric acid was 8.1 ug/dL. Peripheral blood smear showed 3% of blasts and 65% of basophils and bone marrow biopsy revealed marked increase of cellularity. Total cell number of pleural fluid was 14,100/uL and cytological examination of the pleural fluid revealed cells with the morphological features of myeloblasts and occasional megakaryocytes. The bcr-abl gene rearrangement was detected in cells isolated from pleural fluid and bone marrow aspirates. The patient refused treatment with chemotherapeutic for CML and was discharged.
Aged ; Basophils ; Biopsy ; Bone Marrow ; Cell Count ; Dyspnea ; Female ; Gene Rearrangement* ; Granulocyte Precursor Cells ; Hemorrhage ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive* ; Lymph Nodes ; Megakaryocytes ; Pleura ; Pleural Effusion* ; Spleen ; Uric Acid

BACKGROUND: Heat-treated erythrocytes have been reported to be undergone fragmentation and microspherocytes transformation in vitro. However, the changes for enzyme activity of WBCs is not well known. The purpose of this study is to investigate the effects of temperature on function and morphology of WBCs and platelets. And we also defined the relationship between temperature-induced changes in morphology of the erythrocytes and cholesterol level. METHODS: Peripheral blood specimens collected from 55 normal healthy subjects were assayed. CBC and biochemical test for heat-treated specimens were performed. Osmotic fragility was tested and echinocytes were counted using phase contrast microscope. DNA was extracted from heated WBCs with Wizard genomic DNA extraction kit(Promega Corp, USA) and applied on 1.5% agarose gel. After heating, plasma proteins were fractionated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and compared according to changes of temperature. For the phagocytic function of WBCs, 1 x10(7) CFU/mL of gram-negative bacilli were incubated with specimens for 4 hours. Enzymatic activities for myeloperoxidase(MPO), alkaline phosphatase (ALP), and esterase (EST) were confirmed with cytochemical reaction. RESULTS: After incubation at 50 degrees C for 5 min, platelets were counted as 2,978.2 x 10(9)/L and the number of eosinophil was 5.58 x 10(9)/L. And blood glucose level was decreased from 92.4 mg/dL to 59.1 mg/dL, whereas the value of cholesterol was changed as follows; 201.3 mg/dL at 0 sec, 174.6 mg/dL at 60 sec, 151.2 mg/dL at 90 sec, and 197.6 mg/dL at 5 min. In the same temperature, the proportion of echinocytes was 94.1% at 90 sec and decreased to 2.3% at 5 min. In osmotic fragility test, the hemolysis of RBCs began at 0.74% of NaCl and completed at 0.35% of NaCl. Phagocytic function of leukocytes was lost at 52 degrees C, and enzyme activity was lost at following temperature ; MPO at 70t, EST at 70 degrees C, and ALP at 56 degrees C, respectively. SDS-PAGE patterns reveal individual differences of protein at same temperature condition. Aggregating function of platelets were lost, after incubation at 43 degrees C for 5 min. In EDTA-anticoagulated blood, temperature-induced platelelet aggregation was intensified, while in heparinized blood, platelet aggregation was not induced by heat. CONCLUSION: For morphological transformation of erythrocytes in vitro, 50 degrees C is critical point of temperature to induce fragmentation and microspherocytes. Plasma cholesterol appears to play some role to formation of echinocytes induced by heat. MPO is more stable for high temperature than EST and ALP. And sensitivity of RBCs to temperature is considered to be different individually.
Alkaline Phosphatase ; Blood Cells* ; Blood Glucose ; Blood Proteins ; Cholesterol ; DNA ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Eosinophils ; Erythrocytes ; Heating ; Hemolysis ; Heparin ; Hot Temperature* ; Individuality ; Leukocytes ; Osmotic Fragility ; Plasma ; Platelet Aggregation ; Sepharose ; Sodium

BACKGROUND: Serum transferrin receptor (sTfR) level reflects iron status and the rate of erythropoiesis in bone marrow. Iron deficiency still remains one of the most common nutrient deficiency disorders among infants and young children. The purpose of this study is to investigate age-related changes in sTfR level and determine the prevalence of iron deficiency in healthy infant and young children. And we also defined the correlation between iron parameters and sTfR level. METHODS: A total 151 healthy infants and young children aged 4 to 24 months who had been visited Inha University Hospital for vaccination was investigated for the evidence of iron deficiency. The children were divided into 3 groups according to developmental age, i.e., infants aged 4 to 6 months (n=53), infants aged 7 to 12 months (n=37), and children aged 13 to 24 months (n=61). CBC, iron parameters, and sTfR were tested and analyzed. Serum transferrin receptor was assayed by using IDeATMsTfR IEMA (soluble transferrin receptor immunoenzymometic assay, Orion Diagnostica, Orion Co, Finland) test kits. RESULTS: The prevalence of iron deficiency was 26.0% in infants aged 4-6 months, 22.0% in 7-12 months, and 58.3% in 13-24 months. Among 151 subjects, the mean sTfR value in male children was 5.89+/-1.69mg/L and significantly higher than in female children of 5.11+/-1.92mg/L (P=0.019). The level of sTfR in male infants aged 4-6 months was 5.81mg/L and that of female infants with same age was 4.22mg/L (P=0.001). The sTfR level significantly correlated with MCV (r= -0.56, P<0.001), TIBC (r=0.44, P<0.001), and serum iron level (r=-0.42, P<0.001), however correlation between serum iron level and ferritin was poor (r=0.03, P=0.213). CONCLUSION: Iron deficiency still prevail with high incidence in infants and young children especially children aged 13-24 months. The mean value of sTfR is different according to developmental age. Measurement of sTfR appears to be more correlative to iron status than that of ferritin. On the basis of sTfR value, iron need is greater in male infants than in female infants.
Bone Marrow ; Child ; Erythropoiesis ; Female ; Ferritins ; Humans ; Incidence ; Infant* ; Iron* ; Male ; Prevalence ; Receptors, Transferrin* ; Transferrin* ; Vaccination

BACKGROUND: To investigate the change of iron dynamics, erythropoiesis, and serum transferrin receptor (sTfR) concentration during pregnancy, we measured the reticulocyte subpopulations, reticulocyte maturity index (RMI) and sTfR concentration in normal pregnant women. Also we determined which parameter among sTfR, serum iron, or serum ferritin is more representative for clinical features and erythropoiesis in pregnant women. Method: A total of 283 pregnants were examined for reticulocyte subpopulations, sTfR, and a battery of iron parameters. Reticulocytes and their subpopulations were automatically analyzed by flow cytometry (R-3000; Sysmex, Toa, Japan). Serum iron and TIBC were assayed with the automatic chemical analyzer (Hitachi 747; Hitachi, Tokyo, Japan) and serum ferritin was measured by the chemiluminescence method (ACS 180; Chiron, USA). Soluble transferrin receptor was measured by the immunoenzymometric method (ELISA ; Orion Diagnostica, Finland). RESULTS: There was no significant difference in sTfR concentration between pregnant women in the 1st trimester (2.29+/-0.45 mg/L) and non-pregnant women (2.18+/-0.46 mg/L). However, the sTfR concentration gradually increased with gestational age from the 2nd trimester of pregnancy and reached maximal concentration (5.76+/-1.12 mg/L) in the 3rd trimester. The mean sTfR in postpartum 12 weeks was similar to that in the 1st trimester. RMI in the 3rd trimester was three- to four- fold higher than that in the 1st trimester. The sTfR correlated better with RMI (r=0.62, P<0.01), MCH (r=-0.61, P<0.01) and gestational age (r=0.51, P<0.01) than to serum iron and ferritin. CONCLUSIONS: The sTfR concentration increases with gestational age during pregnancy and returns to normal at 12 weeks after delivery. Elevated sTfR concentration during pregnancy seems to be more influenced by erythroid TfR turnover than by iron depletion. The sTfR is a more reliable predictor of erythropoiesis and iron status during pregnancy than serum iron or ferritin.
Erythropoiesis* ; Female ; Ferritins ; Flow Cytometry ; Gestational Age* ; Humans ; Iron ; Luminescence ; Postpartum Period ; Pregnancy ; Pregnant Women* ; Receptors, Transferrin* ; Reticulocytes ; Transferrin*

BACKGROUND: Helicobacter pylori (H. pylori) is closely associated with gastritis, peptic ulcer and gastric carcinoma. We evaluated the reliability and usefulness of 73C-urea breath test (13C-UBT) for the detection of H. pylori infection and searched for the cut-off value of the test. METHOD : We investigated 45 patients, who underwent esophagoduodenoscopy with multiple biopsy specimens taken for culture, histology and rapid urease test, and 13C-UBT. Sensitivity and specificity of UBT were calculated against the combined biopsy-based test results. RESULT: Of 45 patients, 26 were found to be H. pylori-positive according to combined biopsy-based test-results. Sensitivity and specificity of the 13C-UBT were 100.0% and 89.5 %, respectively. CONCLUSION: The urea breath test provides a simple and reliable and noninvasive method of assessing HL pylori infection status.
Biopsy ; Breath Tests* ; Gastritis ; Helicobacter pylori* ; Helicobacter* ; Humans ; Peptic Ulcer ; Sensitivity and Specificity ; Urea* ; Urease

BACKGROUND: Helicobacter pylori (H. pylori) is closely associated with gastritis, peptic ulcer and gastric carcinoma. We evaluated the reliability and usefulness of 73C-urea breath test (13C-UBT) for the detection of H. pylori infection and searched for the cut-off value of the test. METHOD : We investigated 45 patients, who underwent esophagoduodenoscopy with multiple biopsy specimens taken for culture, histology and rapid urease test, and 13C-UBT. Sensitivity and specificity of UBT were calculated against the combined biopsy-based test results. RESULT: Of 45 patients, 26 were found to be H. pylori-positive according to combined biopsy-based test-results. Sensitivity and specificity of the 13C-UBT were 100.0% and 89.5 %, respectively. CONCLUSION: The urea breath test provides a simple and reliable and noninvasive method of assessing HL pylori infection status.
Biopsy ; Breath Tests* ; Gastritis ; Helicobacter pylori* ; Helicobacter* ; Humans ; Peptic Ulcer ; Sensitivity and Specificity ; Urea* ; Urease

Neuroblastoma, which originates in the sympathetic division of the autonomic nervous system, is the most common extracranial solid malignancy in childhood but rarely occurs in adulthood. Only one case of neuroblastoma has been reported in Korean adult. Authors recently experienced a case of neuroblastoma originating from the retroperitoneal cavity with metastasis to the bone marrow and neck. A 29-year-old male was admitted at Inha University Hospital because of abdominal pain and back pain. The patient had a past history of chemotherapy for retroperitoneal tumor in a general hospital 4 years ago. In biochemical test, neuron specific enolase(NSE) was above 260 ng/mL and urine vanillylmandelic acid(VMA) was positive. Immunohistochemical stainings of bone marrow sections, showed positive reactions for NSE, chromogranin, and synaptophysin, however, revealed negative reactions for MIC2, vimentin, and mixed keratin. Pathologic examination showed the characteristic findings of neuroblastoma. We report this case with a review of literature.
Abdominal Pain ; Adult* ; Autonomic Nervous System ; Back Pain ; Bone Marrow* ; Drug Therapy ; Hospitals, General ; Humans ; Male ; Neck ; Neoplasm Metastasis ; Neuroblastoma* ; Neurons ; Synaptophysin ; Vimentin

Autoimmune hemolytic anemia (AIHA) is characterized by the production of Coombs' antibodies, which are responsible for the destruction of RBCs. The antibody specificity of warm AIHA (WAIHA) is very complex while all cells tested are usually reactive in routine test. Although some autoantibodies have broad specificity to the Rh system, apparent specificity for simple Rh antigen (D, C, E, c, e) is rare. A 63 year-old farmer was admitted at Inha University Hospital for evaluation of anemia. He had no history of blood transfusion. He presented overt anemia with decreased Hb (6.6g/dL) and increased reticulocyte count (18.5%, corrected reticulocyte count 8.4%). The direct antiglobulin test (DAT) was strongly positive for IgG and negative for C3d while indirect antiglobulin test (IAT) was weakly positive. Both two different antibody identification test kits identified this antibody as anti-e. His Rh phenotype was CDe. Although the eluted antibody from his own RBCs failed to show specificity against e but agglutinated all the reagent RBCs, we diagnosed this case as WAIHA induced by IgG anti-Rh(e) because his serum agglutinated all e-positive RBCs while it was non-reactive with e-negative RBC in the cross-matching test. Drugs (loxoprofen, cimetidine) couldn't be ruled out as causative agent.
Anemia ; Anemia, Hemolytic, Autoimmune* ; Antibodies ; Antibody Specificity ; Autoantibodies ; Blood Transfusion ; Coombs Test ; Humans ; Immunoglobulin G ; Middle Aged ; Phenotype ; Reticulocyte Count ; Sensitivity and Specificity