BACKGROUND: The binding of some monoclonal antibodies platelet glycoprotein (GP) IIb/IIIa, which is frequently used for flow cytometric immnophenotyping, is known to be inhibited by EDTA. To select the ideal antibodies to be included in the 'Acute Leukemia Panel' for immunophenotyping of acute leukemia, we compared the inhibitory effect of EDTA on the binding of 5 different clones of monoclonal antibodies to platelet GP IIb/IIIa. We also discovered a simple method to neutralize this inhibitory effect. METHODS: Flow cytometric measurement of the number of platelet GP IIb/IIIa binding sites with different anticoagulants was performed using a panel of 5 clones of monoclonal antibodies against CD41 (clone PM6/248), CD41a (clone 96.2C1 & clone HIP8), CD41b (clone HIP2) and CD61 (clone VI-PL2), and the results are expressed as the mean equivalent soluble fluorochrome (MESF) values. RESULTS: The MESF value of the EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody showed a significantly lower value than the MESF of platelets anticoagulated with heparin or citrate (P<0.001). The inhibitory effect of EDTA on the binding of anti-CD41a, clone 96.2C1 antibody to the platelets was neutralized by addition of heparin and CaCl2. The mean MESF value of EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody was significantly increased by the addition of heparin and CaCl2 (P=0.0001). CONCLUSION: The false-negative results of the binding of anti-CD41a, clone 96.2C1 antibody to the platelets seem to be due to the calcium chelating property of EDTA, and the addition of CaCl2 and heparin could be used as an easy compensatory measure for the inhibitory effect of EDTA on other antibodies as well.
Antibodies ; Antibodies, Monoclonal ; Anticoagulants ; Binding Sites ; Blood Platelets ; Calcium ; Citric Acid ; Clone Cells ; Edetic Acid ; Glycoproteins ; Heparin ; Immunophenotyping ; Leukemia

BACKGROUND: The bone marrow biopsy sections of acute leukemia patients occasionally reveal a proliferation of large mononuclear cells that accompany the leukemic blasts, and this proliferation shows a starry sky pattern. We characterized these large mononuclear cells by performing immunohistochemistry with 12 different antibodies. The clinical characteristics were examined and then we determined their difference from hemophagocytic lymphohistiocytosis (HLH) and malignant histiocytic disorders. METHODS: Of the 200 acute leukemic bone marrow biopsy samples, 11 ALL and 10 AML cases showed large mononuclear cell proliferations. The panel of antibodies used for immunohistochemistry included those against the mononuclear phagocyte system, and immunohistochemistry was performed on the patients' initial specimens and the complete remission specimens. 10 normal specimens, 4 initial CML specimens and their complete hematologic response specimens were included as controls. RESULTS: The large mononuclear cells showed immunohistochemical results consistent with histiocytes. They were negative for the markers of dendritic cells the histiocytes and cytokines that are involved in the pathogenesis of HLH and vascular proliferation. Histiocyte proliferation was not observed in the complete remission specimens and in the initial and complete hematological response specimens of the CML patients and the normal bone marrow specimens. None of the cases fulfilled the criteria of HLH, and all 5 ALL cases, for which the immunophenotype results were available, showed a B cell phenotype. CONCLUSION: We characterized the large mononuclear cell proliferations as reactive histiocyte proliferations and we differentiated these from those of secondary HLH and malignant histiocytic disorders. A proportion of the large mononuclear cells showed negative results for all 12 antibodies and they showed characteristics that were suggestive of small fat cells. The pathophysiology and the prognostic effect of the reactive histiocyte proliferation accompanying acute leukemia require further study.
Adipocytes ; Antibodies ; Biopsy ; Bone Marrow ; Cytokines ; Dendritic Cells ; Histiocytes ; Histiocytic Disorders, Malignant ; Humans ; Immunohistochemistry ; Leukemia ; Lymphohistiocytosis, Hemophagocytic ; Mononuclear Phagocyte System ; Phenotype

BACKGROUND: The bone marrow (BM) cellularity has been used as a major index for the evaluation of hematologic diseases and malignancies. However, the microscopic evaluation lacks objectivity because of considerable variations among different observers. We measured myelocrit cellularity as an objective method to evaluate the cellularity. METHODS: Between November 2007 and January 2008, 489 consecutive BM aspirates including 25 cases of AML D7 marrow (7 days after initiation of chemotherapy) were examined. The conventional BM cellularity was evaluated using BM needle biopsies after hematoxylin-eosin stain. EDTA-anticoagulated BM aspirates were put into the Wintrobe tubes, centrifuged and the thickness of 5 layers from the bottom was measured: RBC, buffy coat (BC), plasma, BM particle, and fat layers. The myelocrit cellularity was defined as the ratio of BC to the BC plus fat layers. RESULTS: Both of the thickness of BC layer (r=0.721, P=0.000) and the myelocrit cellularity (r=0.735, P=0.000) correlated well with the conventional cellularity. However, the AML D7 BM cellularity correlated with BC layer (r=0.589, P=0.002), but not with the myelocrit cellularity (r=0.281, P=0.231). The cellularity of the BM other than AML D7 marrow showed a better correlation with the myelocrit cellularity (r=0.826, P=0.000) than the BC layer (r=0.713, P=0.000). CONCLUSIONS: The myelocrit is a simple, reproducible and objective method to determine the BM cellularity. For accurate assessment of BM cellularity, measurement of the thickness of BC layer in AML D7 BM and of the myelocrit cellularity in other BM samples has better be used.
Adolescent ; Adult ; Aged ; Bone Marrow/*pathology ; Cell Separation/instrumentation/*methods ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Leukemia, Myeloid, Acute/*pathology ; Male ; Middle Aged

No abstract available.
BRCA1 Protein/*genetics ; BRCA2 Protein/*genetics ; Base Sequence ; DNA/chemistry ; Databases, Genetic ; Female ; Hereditary Breast and Ovarian Cancer Syndrome/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

BACKGROUND: Automated blood cell analyzers often read leukemic blasts as normal cells. In this study, we evaluated the 5-part differential patterns of blasts using automated analyzers to determine if they can differentiate among blast types. METHODS: Blood samples containing 10% or more blasts were collected from patients with acute leukemia (N=175). The 5-part differential count was conducted using DxH 800 (Beckman Coulter, USA) and XE-2100 analyzers (Sysmex Co., Japan), and the results were compared with manual differential counts, which was used as a reference method. RESULTS: The DxH 800 reported the 5-part white blood cell differential count in 98.9% of the cases. The XE-2100 provided an invalid automated differential count in 72% of the cases. Both analyzers counted most lymphoblasts as lymphocytes and most myeloblasts as monocytes. In 11 cases, the DxH 800 reported a 5-part differential count without a blast flag. CONCLUSIONS: Some automated analyzers are able to recognize and count blasts according to their characteristic cell types. Therefore, complete blood counts obtained automatically can provide valuable data for making provisional decisions regarding the lineage of leukemia cells before further investigation.
Acute Disease ; Automation ; Blood Cell Count/*instrumentation/methods ; Humans ; Leukemia/blood/*diagnosis ; Leukemia, Monocytic, Acute/blood/diagnosis ; Leukemia, Myeloid, Acute/blood/diagnosis ; Leukemia, Promyelocytic, Acute/blood/diagnosis ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood/diagnosis

BACKGROUND: We evaluated the analytical performance of Wako assays for albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), complement C3 and C4, calcium, creatine kinase (CK), C-reactive protein (CRP), direct bilirubin (DBIL), iron, gamma-glutamyl transferase (GGT), HDL cholesterol (HDLC), inorganic phosphorus (IP), LDL cholesterol (LDLC), total bilirubin (TBIL), total protein (TP), and uric acid (UA), as well as the performance of Sekisui assays for albumin, BUN, calcium, CRP, HDLC, IP, LDLC, TP, and UA by using Hitachi LABOSPECT 008 (Hitachi High-Tech Co., Japan). METHODS: Liquid Assayed Multiqual Control (Bio-Rad Laboratories, USA) and pooled patients' sera were analyzed for 20 days. Wako linearity material (Wako Pure Chemical Industries, Ltd., Japan) and Sysmex Interference Check A Plus kit (Sysmex Co., Japan) were used to test linearity and interference, respectively. Concentrations of the target analytes were measured using Hitachi LABOSPECT 008 in 100 residual patient specimens and compared to those in Pureauto S series reagent (Sekisui Medical, Japan), which were measured using Hitachi 7600 (Hitachi High-Tech Co., Japan). RESULTS: Total coefficients of variation (CVs) for the tested analytes were 0.91-9.26% in Wako and 1.04-7.46% in Sekisui assays. Linearity was demonstrated up to the highest concentration within the analytical range in all the assays except for Wako albumin and Sekisui TP. Wako and Sekisui albumin, BUN, CRP, HDLC, and LDLC assays, and in Wako C3, C4, calcium, and UA assays showed no interference with the test concentrations used. All the tested assays, except for Wako AST, LDLC, and TP, and Sekisui calcium and TP, demonstrated comparability with comparative method for at least one medical decision level. CONCLUSIONS: Our study results showed that the analytical performances of Wako and Sekisui chemistry assays evaluated using Hitachi LABOSPECT 008 had appropriate analytical performance for clinical use.
Alanine Transaminase ; Aspartate Aminotransferases ; Bilirubin ; Blood Urea Nitrogen ; C-Reactive Protein ; Calcium ; Chemical Industry ; Chemistry Techniques, Analytical ; Chemistry, Clinical ; Cholesterol, HDL ; Cholesterol, LDL ; Clinical Chemistry Tests ; Complement C3 ; Creatine Kinase ; Humans ; Iron ; Phosphorus ; Transferases ; Uric Acid

BACKGROUND: Most laboratories in Korea have been used kinetic Jaffe method for creatinine measurement. However, kinetic Jaffe method is interfered by hyperbilirubinemia, which causes creatinine decrement. In this study, we evaluated the usefulness of deproteinization by trichloroacetic acid (TCA) in eliminating negative interference of bilirubin for accurate creatinine measurement. METHODS: We evaluated the correction effect of serum creatinine levels by deproteinization using 0.55 mol/L TCA in 43 samples with various total bilirubin levels. For 26 samples of them we measured creatinine using the enzymatic method for evaluating accuracy of TCA correction. Creatinine was measured by using the Toshiba 200-FR automated analyzer and the HiSense CREA reagents. RESULTS: After TCA treatment, 22 to the total 43 samples with more than 10 mg/dL of total bilirubin, revealed statistically higher creatinine concentration (P=0.0002) and the difference of creatinine results is mean 0.53 mg/dL (0.15-1.92 mg/dL). Also, 19 of them (86.4%) revealed 20% or more difference of creatinine results before and after TCA treatment and the negative interference of bilirubin increased in proportion to the rise in total bilirubin concentration (r=0.870). There was no significant difference of creatinine results between kinetic Jaffe method with 0.55 mol/L TCA treatment and enzymatic method (P=0.216). CONCLUSIONS: TCA deproteinization is simple and very efficient method for estimating accurate creatinine level by using kinetic Jaffe method in a patient with hyperbilirubinemia.
Bilirubin ; Creatinine ; Humans ; Hyperbilirubinemia ; Korea ; Trichloroacetic Acid

BACKGROUND: Most laboratories in Korea have been used kinetic Jaffe method for creatinine measurement. However, kinetic Jaffe method is interfered by hyperbilirubinemia, which causes creatinine decrement. In this study, we evaluated the usefulness of deproteinization by trichloroacetic acid (TCA) in eliminating negative interference of bilirubin for accurate creatinine measurement. METHODS: We evaluated the correction effect of serum creatinine levels by deproteinization using 0.55 mol/L TCA in 43 samples with various total bilirubin levels. For 26 samples of them we measured creatinine using the enzymatic method for evaluating accuracy of TCA correction. Creatinine was measured by using the Toshiba 200-FR automated analyzer and the HiSense CREA reagents. RESULTS: After TCA treatment, 22 to the total 43 samples with more than 10 mg/dL of total bilirubin, revealed statistically higher creatinine concentration (P=0.0002) and the difference of creatinine results is mean 0.53 mg/dL (0.15-1.92 mg/dL). Also, 19 of them (86.4%) revealed 20% or more difference of creatinine results before and after TCA treatment and the negative interference of bilirubin increased in proportion to the rise in total bilirubin concentration (r=0.870). There was no significant difference of creatinine results between kinetic Jaffe method with 0.55 mol/L TCA treatment and enzymatic method (P=0.216). CONCLUSIONS: TCA deproteinization is simple and very efficient method for estimating accurate creatinine level by using kinetic Jaffe method in a patient with hyperbilirubinemia.
Bilirubin ; Creatinine ; Humans ; Hyperbilirubinemia ; Korea ; Trichloroacetic Acid

A 32-yr-old male diagnosed with myelodysplastic syndrome underwent an unmanipulated, unrelated, HLA matched, peripheral blood stem cell transplantation. The patient and donor were both blood type O, CcDEe. Twelve weeks post-transplantation, he developed acute autoimmune hemolytic anemia (AIHA). He was transfused multiple times with washed O red cells. High-dose steroid therapy was initiated and he underwent splenectomy; however, AIHA was refractory to therapy. The patient was further treated with combined treatment modalities including immunosuppressive therapy with mycophenolate mofetil and cyclosporine and three cycles of plasma exchange, and AIHA responded to treatment. This is the third case of AIHA complicating hematopoietic stem cell transplantation reported in Korea. Since AIHA is relatively common after hematopoietic stem cell transplantation, accurate and timely diagnosis of the disease and treatment strategies with multiple modalities are necessary.
Adult ; Anemia, Hemolytic, Autoimmune/*diagnosis/drug therapy/etiology ; Combined Modality Therapy ; Cyclosporine/therapeutic use ; Hematopoietic Stem Cell Transplantation/*adverse effects ; Humans ; Male ; Mycophenolic Acid/analogs & derivatives/therapeutic use ; Myelodysplastic Syndromes/complications/diagnosis/therapy ; Plasma Exchange